Rna-guided transcriptional regulation and methods of using the same for the treatment of back pain

ABSTRACT

Described herein are compositions and methods for treatment and prevention of low back pain. The compositions include vectors comprising nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs and a RNA-directed nuclease. The methods include modulating expression of a gene in a cell using said compositions, introducing a CRISPR-Cas system into a cell comprising one or more vectors comprising said compositions, inducing site-specific DNA cleavage in a cell, and treating a subject having lower back pain, and lower back pain caused by degenerative disc disease using the compositions disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Application 62/230,931, which was filed on Jun. 18, 2015.The content of this earlier filed application is hereby incorporated byreference herein in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant numbersR03AR068777 and F32 AR063012 awarded by National Institutes of Health.The government has certain rights in the invention.

INCORPORATION OF THE SEQUENCE LISTING

The present application contains a sequence listing that was submittedin ASCII format via EFS-Web concurrent with the filing of theapplication, containing the file name SL_21101_0331P1 which is 17,221bytes in size, created on Jun. 16, 2016, and is herein incorporated byreference in its entirety.

BACKGROUND

Lower back pain (LBP) is the single leading cause of disabilityworldwide having a global lifetime prevalence of 38.9%. Degenerativedisc disease (DDD) and associated pathologies are considered majorcontributors to LBP. The progression of DDD is associated with aninflammatory environment that includes the presence of inflammatorycytokines (e.g., TNF-α, IL-Iβ) in the intervertebral disc (IVD) that areactive in the degenerative process and may sensitize pain-sensing nervefibers in the IVD.

Although both surgical and non-surgical treatments for DDD-induced LBPare able to alleviate symptoms, they, however, fail to prevent theprogression of disc degeneration, thus, LBP often returns aftertreatment. To effectively treat DDD-induced LBP on a long-term basis,therapeutic methods that can slow DDD progression and reduce the needfor surgical intervention are needed. DDD and its progression have beenassociated with the action of inflammatory cytokines in theintervertebral disc (IVD) that signal the breakdown of the extracellularmatrix through their respective receptors. Therefore a method foreffectively slowing DDD progression that inhibits the catabolicsignaling of these inflammatory cytokines in the IVD is also needed.

SUMMARY

Disclosed herein, are CRISPR-Cas systems comprising one or more vectorscomprising: a) a promoter operably linked to one or more nucleotidesequences encoding a CRISPR-Cas system guide RNA (gRNA), wherein thegRNA hybridizes with a target sequence of a DNA locus in a cell; and b)a regulatory element operably linked to a nucleotide sequence encoding aRNA-directed nuclease, wherein components a) and b) are located on thesame or different vectors of the same system, wherein the gRNA targetsand hybridizes with the target sequence and directs the RNA-directednuclease to the DNA locus; and wherein the gRNA sequence is selectedfrom the group listed in Table 2 and Table 4.

Disclosed herein, are vectors comprising promoters operably linked toone or more nucleotide sequences encoding a CRISPR-Cas system guide RNA(gRNA); and regulatory elements operably linked to a nucleotide sequenceencoding a RNA-directed nuclease; wherein the gRNA sequence is selectedfrom the group listed in Table 2 and Table 4.

Disclosed herein, are methods of modulating of genes in cells, themethods comprising: introducing into the cells a first nucleic acidencoding a guide RNA (gRNA), wherein the gRNA comprises a DNA-bindingdomain, wherein the nucleic acid is operably linked to a regulatoryelement, wherein the gRNA is complementary to a target nucleic acidsequence comprising the gene; introducing into the cell a second nucleicacid encoding a transcriptional regulator protein or domain thatmodulates the target nucleic acid expression, and comprises agRNA-binding domain, wherein the second nucleic acid is operably linkedto a regulatory element; and introducing into the cell a third nucleicacid encoding a deactivated nuclease Cas9 (dCas9) protein, wherein thethird nucleic acid is operably linked to a regulatory element, whereinthe dCas9 protein interacts with the gRNA and is fused to thetranscriptional regulator protein; wherein the cell produces the gRNAthat binds the dCas9 protein and the transcriptional regulator proteinor domain fused to the DNA-binding domain; wherein the guide RNA and thedCas9 protein co-localize to the target nucleic acid sequence andwherein the transcriptional regulator protein or domain modulatesexpression of the gene; wherein the gRNA sequence is selected from thegroup listed in Table 2 and Table 4.

Disclosed herein, are methods for introducing into a cell a CRISPR-Cassystem comprising one or more vectors, the method comprising: a promoteroperably linked to one or more nucleotide sequences encoding aCRISPR-Cas system guide RNA (gRNA), wherein the gRNA hybridizes with atarget sequence of a DNA molecule in a cell; a regulatory elementoperably linked to a nucleotide sequence encoding a RNA-directednuclease, wherein components a) and b) are located on the same ordifferent vectors of the same system, wherein the gRNA targets andhybridizes with the target sequence and directs the RNA-directednuclease to the DNA molecule; wherein the gRNA sequence is selected fromthe group listed in Table 2 and Table 4.

Disclosed herein, are methods for introducing into a cell a vectorcomprising: a promoter operably linked to one or more nucleotidesequences encoding a CRISPR-Cas system guide RNA (gRNA); a regulatoryelement operably linked to a nucleotide sequence encoding a RNA-directednuclease; wherein the gRNA sequence is selected from the group listed inTable 2 and Table 4.

Disclosed herein, are methods of treating a subject having degenerativedisc disease, the method comprising: (a) determining the subject hasdegenerative disc disease; and (b) administering to the subject apharmaceutical composition comprising a nucleic acid sequence encoding aCRISPR-associated deactivated endonuclease and one or more guide RNAs,wherein the guide RNA is selected from the group listed in Table 2 andTable 4.

Other features and advantages of the present compositions and methodsare illustrated in the description below, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E show DRG neuron imaging. FIG. 1A is a representative calciumsignal (ΔF/F) for a neuron firing in response to heat stimuli. DRGneurons were considered to have fired when the ΔF/F for the neuronexceeded the threshold level defined as the mean ΔF/F value at baselineplus three standard deviations. FIG. 1B shows calcium images ofrepresentative DRG neurons at baseline, immediately following heatapplication (FIG. 1C), at maximum response to heat stimuli (FIG. 1D),and return to baseline following stimuli (FIG. 1E).

FIGS. 2A-C show degenerative IVDs sensitize peripheral neurons toheating. FIG. 2A shows the firing response of rat DRG neurons as afunction of temperature was fitted to a Boltzmann curve for thedegenerative IVD conditioned media (n=5 patients), healthy IVDconditioned media (n=3) patients), and control media groups. The T50(FIG. 2B) and maximum (FIG. 2C) generated from dose response curvefitting for the degenerative IVD conditioned media, healthy IVDconditioned media, and control media groups. *=p<0.05 when compared tothe control media group for the same temperature. #=p<0.05 when comparedto the healthy IVD conditioned media group for the same temperature.

FIGS. 3A-D show that IL-6 is the primary mediator of peripheral neuronsensitization in the degenerative IVD. FIG. 3A shows the mean Boltzmanncurve fittings of neuron response to thermal stimuli for the controlmedia, conditioned media plus IL-6 antibody, conditioned media, andconditioned media plus isotype control antibody groups. The maximum(FIG. 3B) and T50 (FIG. 3C.) generated from dose response curve fittingfor the control media, conditioned media plus IL-6 antibody (20 μg/mL),conditioned media plus isotype control antibody (20 μg/mL), andconditioned media groups (n=5). FIG. 3D shows the percentage of DRGneurons firing in response to temperatures of 39° C. and 44° C. whenexposed to conditioned media or conditioned media plus IL-6 antibody.FIG. 3A-C, Values are mean±standard deviations. N=5 for all groupstested. *=p<0.05 compared to the control media group at the sametemperature. #=p<0.05 compared to the conditioned media plus IL-6antibody group at the same temperature. FIG. 3D is table showing thevalues as a percentage of neurons firing in response to thermal stimuli.*=p<0.05 compared to the conditioned media plus IL-6 antibody group forthe same patient at the same temperature.

FIG. 4 shows that AKAP mediates sensitization of DRG neurons to heatingin the degenerative IVD. Values are the mean±standard deviations.*=p<0.01 compared to the control media group. #=p<0.01 when compared tothe degenerative IVD conditioned media plus St-Ht31P group.

FIG. 5 shows degenerative IVD conditioned media and acidic pHsynergistically enhance sensory neuron desensitization to heating andtrigger spontaneous firing.

FIGS. 6A-G show epigenomic editing of AKAP 150 expression in DRG Neuronsabolishes degenerative IVD induced neuron sensitization to heating. FIG.6A shows CRISPR based epigenomic editing vectors contains sgRNAs thatdirects the dCas9 endonuclease fused with KRAB to the PAM bindingsequence of the genomic DNA leading to the expression of KRAB. FIG. 6Bshows gene expression occurs when chromatin is maintained in theeuchromatin (open) configuration by acetylation of the H3K9 histones.Expression of KRAB recruits endogenous factors that replace acetylationof H3K9 with tri-methylation, maintaining chromatin in theheterochromatic state, silencing gene expression. FIG. 6C shows thevector map of CRISPR epigenome editing targeting the AKAP 150 gene. FIG.6D shows epigenome editing of AKAP 150 expression in rat DRG neurons.FIG. 6E shows the mean Boltzman fit curves of neuron firing response tothermal stimuli for naïve neurons exposed to control media and naïvecells, AKAP epigenome edited cells and non-target epigenomically editedneurons exposed to degenerative IVD conditioned media. The T50 (FIG. 6F)and maximum response (FIG. 6G) generated from the dose response curvefrom naïve neurons exposed to control media and naïve cells, AKAPepigenome edited cells and non-target epigenomically edited neuronsexposed to degenerative IVD conditioned media.

FIGS. 7A-D show the results of screening for efficient epigenome editingin HEK293T cells. FIG. 7A shows the lentiviral cassette demonstratingcomponents of the epigenome editing system and how their expression isdriven. FIG. 7B is a bar graph showing the screening of gRNAs for TNFR1significantly downregulated by 4 gRNAs. FIG. 7C is a bar graph showingthe screening of gRNAs for IL1R1 significantly downregulated by 2 gRNAs.(* denotes p<0.05 compared to nontarget control). FIG. 7D shows a foldchange in NF-κB activity in response to TNF-α dosing in HEK293T cellswith TNFR1 edit mediated by gRNA 1 (*=p<0.05 (TNFR1/IL1R1 Edit vs.Naïve), #=p<0.05 (TNF-α/IL-1β dose vs. no dose control)).

FIGS. 8A-B show lentiviral mediated gene and receptor signalingdownregulation in hADMSCs. FIG. 8A shows TNFR1 and IL1R1 expression inhADMSCs post-transduction of epigenome editing system under the controlof selected gRNAs. FIG. 8B shows changes in NF-κB activity postTNF-α/IL-1β dosing in epigenome edited hADMSCs which express theepigenome editing system with the most efficient guides for eachreceptor (*=p<0.05 (TNFR1/IL1R1 Edits vs. nontarget Edit), #=p<0.05(TNF-α/IL-1β dose vs. no dose control)).

FIGS. 9A-F show cell proliferation and ECM deposition in 3D culture byepigenome edited cells in the presence of cytokines. FIGS. 9A and 9Bshow the cross sectional area of cell pellets in mm², cultured with orwithout TNF-α/IL-1β (n=6). FIGS. 9C and 9D show percent changes in DNAcontent relative to undosed control (n=4-5). FIG. 9E is a representativeimage of hADMSC pellets, demonstrating visible size differences betweenTNFR1/IL1R1 edit cells and non-target edit control under inflammatoryconditions. FIG. 9F shows H&E staining of cell pellets visualizingrelative ECM content show lighter ECM staining in dosed non-target editcells but not in TNFR1/IL1R1 edited cells. (* denotes p<0.05 compared todosed nontarget edit control).

FIGS. 10A-F show the results of quantification of GAG contents in pelletcultures that have undergone chondrogenic differentiation for 3 weekswith or without the presence of TNF-α/IL-1β. FIGS. 10A and 10B show GAGcontent per pellet (n=5). FIGS. 10C and 10D) show the amount of GAGreleased into media during culture (n=7-8). FIGS. 10E and 10F show thesum of GAG content that was released and within cell pellets (n=5).(*=p<0.05 relative to 0 ng/mL control, #=p<0.05 relative to dosed TNFR1edited cells.)

FIGS. 11A-B show the ability of epigenome edited hADMSCS to suppressPBMC proliferation in coculture. FIG. 11A shows the fold change inproliferation relative to PBMCs alone demonstrate suppression of PBMCproliferation by all hADMSCs (n=4). FIG. 11B shows a representative flowcytometry graph of PBMCs demonstrating a visible decrease in CD45 EdUpositive cells (graphs represent condition they are directly beneath inpart A graph). (*=p<0.05 relative to PBMCs alone, #=p<0.05 relative tococulture with naïve hADMSCs.)

FIGS. 12A-B show knockdown of TNFR1 (A) and IL1R1 (B) with multiplepromoter-targeting gRNAs (*=P<0.05 compared to non-target control).

FIGS. 13A-B show transduced primary human NP cells (A) expressingCRISPRi system as evidenced by GFP expression and the knockdown of TNFR1in NP cells using TNFR1 CRISPRi lentiviral vector (B).

FIG. 14 shows the fold change in NF-KB activity by HEK293T cells dosedwith TNF-α. Differences between the knockdown and the number ofknockdown cells are statistically significant (P<0.05) for each dose asdenoted by *.

FIG. 15 shows an example of a singleplex upregulation vector. The vectorshown comprises dCas9-p300core.

FIG. 16 shows an example of a singleplex upregulation vector. The vectorshown comprises dCas9-KRAB.

FIG. 17 shows an example of a multiplex upregulation vector. The vectorshown comprises dCas9-p300core.

FIG. 18 shows an example of a multiplex upregulation vector. The vectorshown comprises dCas9-KRAB.

DETAILED DESCRIPTION

Many modifications and other embodiments of the present disclosure setforth herein will come to mind to one skilled in the art to which thisdisclosure pertains having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the present disclosure is not to be limited to thespecific embodiments disclosed and that modifications and otherembodiments are intended to be included within the scope of the appendedclaims. Although specific terms are employed herein, they are used in ageneric and descriptive sense only and not for purposes of limitation.

Before the present compositions and methods are disclosed and described,it is to be understood that they are not limited to specific syntheticmethods unless otherwise specified, or to particular reagents unlessotherwise specified, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentdisclosure, example methods and materials are now described.

Moreover, it is to be understood that unless otherwise expressly stated,it is in no way intended that any method set forth herein be construedas requiring that its steps be performed in a specific order.Accordingly, where a method claim does not actually recite an order tobe followed by its steps or it is not otherwise specifically stated inthe claims or descriptions that the steps are to be limited to aspecific order, it is in no way intended that an order be inferred, inany respect. This holds for any possible non-express basis forinterpretation, including matters of logic with respect to arrangementof steps or operational flow, plain meaning derived from grammaticalorganization or punctuation, and the number or type of aspects describedin the specification.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present disclosure is not entitled to antedate such publicationby virtue of prior disclosures. Further, the dates of publicationprovided herein can be different from the actual publication dates,which can require independent confirmation.

Definitions

As used in the specification and in the claims, the term “comprising”can include the aspects “consisting of” and “consisting essentially of.”“Comprising can also mean “including but not limited to.”

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” can include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a compound”includes mixtures of compounds; reference to “a pharmaceutical carrier”includes mixtures of two or more such carriers, and the like.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance may or may not occur andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “sample” is meant a tissue or organ from asubject; a cell (either within a subject, taken directly from a subject,or a cell maintained in culture or from a cultured cell line); a celllysate (or lysate fraction) or cell extract; or a solution containingone or more molecules derived from a cell or cellular material (e.g. apolypeptide or nucleic acid), which is assayed as described herein. Asample may also be any body fluid or excretion (for example, but notlimited to, blood, urine, stool, saliva, tears, bile) that containscells or cell components.

As used herein, the term “subject” refers to the target ofadministration, e.g., a human. Thus the subject of the disclosed methodscan be a vertebrate, such as a mammal, a fish, a bird, a reptile, or anamphibian. The term “subject” also includes domesticated animals (e.g.,cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats,etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig,fruit fly, etc.). In one aspect, a subject is a mammal. In anotheraspect, a subject is a human. The term does not denote a particular ageor sex. Thus, adult, child, adolescent and newborn subjects, as well asfetuses, whether male or female, are intended to be covered.

As used herein, the term “patient” refers to a subject afflicted with adisease or disorder. The term “patient” includes human and veterinarysubjects. In some aspects of the disclosed methods, the “patient” hasbeen diagnosed with a need for treatment for an infectious disease, suchas, for example, prior to the administering step.

Ranges can be expressed herein as from “about” or “approximately” oneparticular value, and/or to “about” or “approximately” anotherparticular value. When such a range is expressed, a further aspectincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” or “approximately,” it will be understood thatthe particular value forms a further aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein and that each value is also herein disclosed as “about”that particular value in addition to the value itself. For example, ifthe value “10” is disclosed, then “about 10” is also disclosed. It isalso understood that each unit between two particular units is alsodisclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and14 are also disclosed.

“Inhibit,” “inhibiting” and “inhibition” mean to diminish or decrease anactivity, response, condition, disease, or other biological parameter.This can include, but is not limited to, the complete ablation of theactivity, response, condition, or disease. This may also include, forexample, a 10% inhibition or reduction in the activity, response,condition, or disease as compared to the native or control level. Thus,in an aspect, the inhibition or reduction can be a 10, 20, 30, 40, 50,60, 70, 80, 90, 100%, or any amount of reduction in between as comparedto native or control levels. In an aspect, the inhibition or reductionis 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% ascompared to native or control levels. In an aspect, the inhibition orreduction is 0-25, 25-50, 50-75, or 75-100% as compared to native orcontrol levels.

“Modulate”, “modulating” and “modulation” as used herein mean a changein activity or function or number. The change may be an increase or adecrease, an enhancement or an inhibition of the activity, function ornumber.

“Promote,” “promotion,” and “promoting” refer to an increase in anactivity, response, condition, disease, or other biological parameter.This can include but is not limited to the initiation of the activity,response, condition, or disease. This may also include, for example, a10% increase in the activity, response, condition, or disease ascompared to the native or control level. Thus, in an aspect, theincrease or promotion can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%,or more, or any amount of promotion in between compared to native orcontrol levels. In an aspect, the increase or promotion is 10-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared tonative or control levels. In an aspect, the increase or promotion is0-25, 25-50, 50-75, or 75-100%, or more, such as 200, 300, 500, or 1000%more as compared to native or control levels. In an aspect, the increaseor promotion can be greater than 100 percent as compared to native orcontrol levels, such as 100, 150, 200, 250, 300, 350, 400, 450, 500% ormore as compared to the native or control levels.

As used herein, the term “determining” can refer to measuring orascertaining a quantity or an amount or a change in activity. Forexample, determining the amount of a disclosed polypeptide in a sampleas used herein can refer to the steps that the skilled person would taketo measure or ascertain some quantifiable value of the polypeptide inthe sample. The art is familiar with the ways to measure an amount ofthe disclosed polypeptides and disclosed nucleotides in a sample.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA or aDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids as disclosed herein can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid or thiodiesterlinkages). In particular, nucleic acids can include, without limitation,DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

As used herein, the term “complementary” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick or other non-traditional types. Apercent complementary indicates the percentage of residues in a nucleicacid molecule which can form hydrogen bonds (e.g., Wastson-Crick basepairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).

As used herein, the term “vector” or “construct” refers to a nucleicacid sequence capable of transporting into a cell another nucleic acidto which the vector sequence has been linked. The term “expressionvector” includes any vector, (e.g., a plasmid, cosmid or phagechromosome) containing a gene construct in a form suitable forexpression by a cell (e.g., linked to a transcriptional control elementor regulatory element). The terms “plasmid” and “vector” can be usedinterchangeably, as a plasmid is a commonly used form of vector.Moreover, this disclosure is intended to include other vectors whichserve equivalent functions.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

Low back pain is the leading cause of disability worldwide[58], ranksthird in disease burden according to disease adjusted life years[40],and generates a tremendous socio-economic cost [26]. Numerous factorshave been associated with back pain, including degenerative discdisease, which is characterized by the breakdown of the intervertebraldisc (IVD) extracellular matrix (ECM)[35,48], a loss of disc height[55],an inflammatory response [9,10,16,31,39,52,53,57], and alteredinnervation of the IVD[14,16,17]. Despite the observation of thesechanges in the degenerative IVD and hypotheses on the relationship ofthese changes to painful symptoms[18,28,34,47], the underlyingmechanisms are not well understood and treatment strategies are limited.Described herein is a model that was developed to demonstrate theunderlying sensitizing interactions between the degenerative disc andperipheral neurons and used to demonstrate targeted clusteredregularly-interspaced palindromic repeat (CRISPR) epigenome editing tomodulate these degenerative IVD induced sensitivities.

In the healthy IVD, neurons innervate the outer lamellae of the IVD andoriginate in the dorsal root ganglion (DRG). The majority of theseneurons are nociceptive neurons expressing calcitonin gene-relatedpeptide (CGRP)[2-4,29,43] and TRPV1[2,4,39,43]. In degenerative IVDs,the number of nociceptive neurons innervating the disc increases[25] andnociceptive neurons expressing CGRP[4,7] extend into typically aneuralregions of the inner AF and NP[4,7,14,16,17,33]. Nociceptive neuronsinnervating the degenerative IVD are exposed to pathologically highlevels of IL-6, TNF-α, and IL-1β[9,11,36,37,52] and to pathologicallylow pH levels[30]. TNF-α, IL-1β, and IL-6 have been demonstrated tosensitize nociceptive neurons to heating[41,42,45] and induce thermalhyperalgesia[1,15,44,45] in models of peripheral neuropathy.Additionally, acidic pH (e.g., 6.0-7.0) lowers the temperature thresholdof TRPV1 and potentiates signaling through TRPV1[12]. As a result, thepresence of multiple sensitizing factors in the degenerative IVD maytrigger discogenic pain by sensitizing TRPV1 to stimuli that arenon-painful in healthy patients. Described herein is an in vitro modeldeveloped to investigate these interactions and test CRISPR epigenomeediting strategies in peripheral neurons to regulate these interactions.

CRISPR epigenome editing allows for stable, site-specific[56] epigenomemodifications to modulate gene expression. Briefly, CRISPR-Cas9-basedepigenome editing utilizes a nuclease-deficient Cas9 (dCas9) and asynthetic guide RNA to target specific DNA sequences[13,24,38]. Thefusion of KRAB to dCas9 produces targeted H3K9 methylation[20,32,46,54],which can be used to regulate endogenous gene expression. Disclosedherein are compositions and methods for direct regulation of peripheralneuron sensitization via CRISPR epigenome editing to treat, for example,discogenic back pain symptoms. Using this technique, back pain may betreated by epigenome modifications of pain related genes in nociceptiveneurons.

Disclosed herein are models developed to test the hypothesis thatdegenerative IVD conditions (e.g., inflammatory cytokines and acidic pH)can induce sensitization of nociceptive neurons to noxious stimuli andto demonstrate CRISPR epigenomic editing of nociceptive neurons as apotential discogenic back pain treatment by regulating the peripheralneuron response to these deleterious interactions. This study elucidatesthe synergistic effects of low pH and the IL-6/AKAP/TRPV1 pathway asresponsible for degenerative IVD neuron sensitization and demonstratesepigenomic regulation of this pathway as a pain modulation strategy.

Low back pain (LBP) is a widespread problem, ranking first overall inyears lived with disability (Murray and Lopez, 2013), and having anestimated global lifetime prevalence of 38.9% (Hoy et al., 2012).Degenerative disc disease (DDD) is considered a major contributor to LBP(Luoma et al., 2000). Currently both surgical and non-surgicaltreatments for DDD induced LBP are able to alleviate symptoms but theydon't provide a mechanism for preventing the progression of discdegeneration, thus, often LBP may return after treatment (Von Korff andSaunders, 1996). In order to effectively treat DDD induced LBP on along-term basis, therapeutic methods that can slow DDD progression andreduce the need for surgical intervention are needed. Regarding DDD, itsprogression has been associated with the action of inflammatorycytokines in the intervertebral disc (IVD) that signal the breakdown ofthe extracellular matrix (ECM) through their respective receptors(Millward-Sadler et al., 2009; Studer et al., 2011; Purmessur et al.,2013). Therefore a potential method for effectively slowing DDDprogression could be to inhibit the catabolic signaling of theseinflammatory cytokines in the IVD.

Inhibition of catabolic signaling by inflammatory cytokines may be doneby delivering mesenchymal stem cells (MSCs) that are known to havetherapeutic immunomodulatory properties (Wang et al., 2014). Delivery ofMSCs for treatment of DDD has shown efficacy both pre-clinically andclinically in decreasing pain and/or promoting IVD tissue regeneration(Orozco et al., 2011; Marfia et al., 2014; Pettine et al., 2016; Chun etal., 2012). These therapeutic cells are believed to provide regenerativeeffects mostly by stimulating anabolic gene expression through paracrinesignaling (Strassburg et al., 2010; Tam et al., 2014). This is useful inproviding a short term regenerative effect but the long term effects ofthis mechanism of action are unclear. In some aspects, in order toprovide long term effects delivered MSCs must remain viable in the IVD.This likely requires them to differentiate into nucleus pulposus (NP) orchondrocyte like cells in order to withstand the low pH, high osmolarityenvironment of the IVD (Wuertz et al., 2008; Liang et al., 2012). Withincreased levels of inflammatory cytokines TNF-α and IL-1β known to bein the degenerative IVD (Le Maitre et al., 2007; Weiler et al., 2005)differentiation may be inhibited (Wehling et al., 2009; Heldens et al.,2012). Therefore to promote survival and regeneration by implanted MSCsin the IVD, it may be beneficial to regulate signaling of TNF-α andIL-1β in MSCs to be delivered to the degenerative IVD.

To regulate signaling of TNF-α and IL-1β, binding to their respectivereceptors must be inhibited. This may be achieved through cytokinespecific inhibitors delivered directly or by gene therapy, but use ofinhibitors has drawbacks. Regarding direct delivery, continuous deliveryis needed for long-term inhibition due to the short half-life of theinhibitor molecules. Regarding both types of delivery, the inhibitorsaren't cell or receptor specific, thus, they may inhibit any pathwaythat TNF-α and IL-1β signals, on any cell presenting the appropriatereceptors within their vicinity. A more controllable method forinhibition of signaling is regulating the presence of the particularreceptors as signal transducing receptors act upon the cells they arepresented on and regulate specific pathways. Being able to regulatespecific pathways is important as not all functions of theseinflammatory cytokines are negative. For example, concerning TNF-αreceptor signaling through TNFR1 can result in either apoptotic oranti-apoptotic signaling but signaling through TNFR2 is known to resultin anti-apoptotic pathways, thus, it is of interest to specificallytarget TNFR1 signaling (Cabal-Hierro and Lazo, 2012).

To decrease the presence of specific inflammatory cytokine receptors,one must regulate their protein or gene expression for which there areseveral methods available. A recently developed method of generegulation at the genomic level, Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) based epigenome editing (Thakore et al.,2015; Larson et al., 2013), is of interest for regulating receptorexpression and therefore signaling of inflammatory cytokines. It hasbeen shown to perform highly specific and effective gene modulation inmammalian cells and has been shown to be more robust in downregulatingexpression compared to RNAi (Gilbert et al., 2014). A study describedherein aimed to investigate the functional effects of regulatingexpression of TNFR1 and IL1R1, via CRISPR based epigenome editing. Thiswas first screened in HEK293T cells to allow for rapid design, and thenmore thoroughly investigated in immortalized human adipose derivedmesenchymal stem cells (hADMSCs). These hADMSCs were used as theyprovide an in vitro model for a clinically relevant cell populationregarding cell delivery to the degenerative IVD. It was hypothesizedthat downregulation of TNFR1 and IL1R1 by epigenome editing will inhibitinflammatory signaling that results in ECM degradation, cell apoptosis,and inhibition of differentiation therefore engineering cells that aremore likely to have a regenerative effect within the environment of thedegenerative IVD once implanted.

The newly developed tool for endogenous gene regulation, CRISPRinterference (CRISPRi), has the potential to provide effective multiplexgene regulation for applications in DDD. It has been shown that CRISPRican perform specific and effective gene knockdown in mammalian cells ina single or multiplex manner [3]. As described herein, this systemrequires the expression of nuclease-inactive Cas9 (dCas9) by itself orfused to the Kruppel-associated box (KRAB) and the expression of shortgenomic loci-specific guide RNAs (gRNAs) that are complementary to thepromoter of the gene [4]. As further disclosed herein, the CRISPRisystem can be used to perform multiplex knockdown of inflammatorycytokine receptors and inhibit actions of inflammatory cytokines oncells in the IVD and retard the progression of DDD. Herein, themodulation of TNFR1 and IL1R1 using CRISPRi in both HEK293T cells andhuman nucleus pulpous cells and the ability to alter cell response toinflammatory challenge is demonstrated.

Described herein are compositions and methods directed to CRISPRiregulation of inflammation in the intervertebral disc and nervous systemfor the treatment of back pain related pathologies. The compositionsdescribed herein can be delivered directly to one or more intervertebraldiscs, for example, after a discectomy procedure to halt the progressionof disc degeneration after surgery; delivered to adjacent intervertebraldiscs, for example, after spinal fusion surgery to halt the progressionof disc degeneration (e.g., adjacent segment disease) after surgery;delivered to one or more peripheral nerves to, for example, antagonizesensitization of nociceptive neurons due to inflammatory signaling forthe treatment of neuropathies, including but not limited toradiculopathy and discogenic back pain; and delivered to the centralnervous system, for example, to antagonize altered neuronal signalingdue to inflammatory signaling for the treatment of neuropathies,including but not limited to radiculopathy and discogenic back pain.

Compositions

The compositions disclosed herein include a CRISPR-Cas system. TheCRISPR-Cas system can be non-naturally occurring. In some aspects, theCRISPR-Cas system comprises one or more vectors. In some aspects, thevectors can be singleplex or multiplex vectors. For example, vectorsthat can be used in the disclosed compositions and methods can include,but are not limited to the vectors shown in FIGS. 15-18. In an aspect, asingleplex vector is a repression vector. In an aspect, a singleplexvector is an upregulation vector. In an aspect, a multiplex vector is arepression vector. In an aspect, a multiplex vector is an upregulationvector. In an aspect, the vector is a combination repression,upregulation vector.

In some aspects, the one or more vectors comprise a promoter operablylinked to one or more nucleotide sequences encoding a CRISPR-Cas systemguide RNA (gRNA). In some aspects, the gRNA can hybridize with a targetsequence of a DNA molecule or locus in a cell. In some aspects, the oneor more vectors can also comprise a regulatory element operably linkedto a nucleotide sequence encoding a RNA-directed nuclease. In someaspects, the promoter operably linked to one or more nucleotidesequences encoding a CRISPR-Cas system gRNA and the regulatory elementoperably linked to a nucleotide sequence encoding a RNA-directednuclease can be located on the same or different vectors of the samesystem. The gRNA can target and hybridize with the target sequence. Insome aspects, the gRNA can also direct the RNA-directed nuclease intothe DNA molecule or locus. In some aspects, gRNA can be selected fromthe group listed in Tables 2 and 4.

As used herein, the term “regulatory element” refers to promoters,promoter enhancers, internal ribosomal entry sites (IRES) and otherelements that are capable of controlling expression (e.g., transcriptiontermination signals, including but not limited to polyadenylationsignals and poly-U sequences). Regulatory elements can directconstitutive expression. Regulatory elements can be tissue-specific.Examples of tissue-specific promoters can direct expression in a desiredtissue of interest (e.g., muscle, neuron, bone, skin, blood,intervertebral disc), specific organs (e.g., liver, pancreas, brain,spinal cord), or particular cell types (peripheral nerves, annulusfibrosus, nucleus pulposus, chondrocytes). Regulatory elements can alsodirect expression in a temporal-dependent manner including but notlimited to cell-cycle dependent or developmental stage-dependent.Temporal-dependent expression can be tissue or cell-type specific.Regulatory element can also refer to enhancer elements. Examples ofenhancer elements include but are not limited to WPRE, CMV enhancers,and SV40 enhancers. In an aspect, the regulatory element is hUbC. In anaspect, the hUbC promoter is operably linked to a nucleotide sequenceencoding a RNA-directed nuclease. Generally, any constitutive promotercan be operably linked to a nucleotide sequence encoding a RNA-directednuclease. Specific gene specific promoters can be used. Such promotersallow cell specific expression or expression tied to specific pathways.Any promoter that is active in mammalian cells can be used. In anaspect, the promoter is an inducible promoter including, but not limitedto, Tet-on and Tet-off systems. Such inducible promoters can be used tocontrol the timing of the desired expression.

The transcriptional control element can be a promoter. In an aspect, thepromoter can be a mammalian cell active promoter (e.g., SV40, CMV, SP6,T7); a yeast active promoter (e.g., GAL4); a bacteria active promoter(e.g., Lac); or a baculovirus/insect cell active promoter (e.g.,polyhedron). In an aspect, the transcriptional control element can be aninducible promoter. Examples of inducible promoters include but are notlimited to tetracycline inducible system (tet); heat shock promoters andIPTG activated promoters.

Disclosed herein, are vectors comprising a promoter operably linked toone or more nucleotide sequences encoding a CRISPR-Cas system gRNA and aregulatory element operably linked to a nucleotide sequence encoding aRNA-directed nuclease. In some aspects, the gRNA sequence can beselected from the group listed in Table 2 and Table 4. In some aspects,the promoter operably linked to one or more nucleotide sequencesencoding a CRISPR-Cas system gRNA and a regulatory element operablylinked to a nucleotide sequence encoding a RNA-directed nuclease can beon the same or different vectors of the same system.

Vectors include, but are not limited to nucleic acid molecules that aresingle-stranded double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a viral vector, wherein virally-derived DNA or RNA sequences arepresent in the vector for packaging into a virus. Viral vectors caninclude polynucleotides carried by a virus for transfection into a hostcell. In some aspects, the CRISPR-Cas system described herein ispackaged into a single lentiviral, adenoviral or adeno-associated virusparticle.

The vectors disclosed herein can also include detectable labels. Suchdetectable labels can include a tag sequence designed for detection(e.g., purification or localization) of an expressed polypeptide. Tagsequences include, for example, green fluorescent protein, glutathioneS-transferase, polyhistidine, c-myc, hemagglutinin, or Flag™ tag, andcan be fused with the encoded nucleic acid.

Some vectors are capable of autonomous replication in a host cell whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and are thereby replicatedalong with the host genome.

The term “expression vector” is herein to refer to vectors that arecapable of directing the expression of genes to which they areoperatively-linked. Common expression vectors of utility in recombinantDNA techniques are often in the form of plasmids. Recombinant expressionvectors can comprise a nucleic acid as disclosed herein in a formsuitable for expression of the acid in a host cell. In other words, therecombinant expression vectors can include one or more regulatoryelements or promoters, which can be selected based on the host cellsused for expression that is operatively linked to the nucleic acidsequence to be expressed.

The term “operatively linked to” refers to the functional relationshipof a nucleic acid with another nucleic acid sequence. Promoters,enhancers, transcriptional and translational stop sites, regulatoryelements, regulatory control elements and other signal sequences areexamples of nucleic acid sequences operatively linked to othersequences. For example, operative linkage of DNA to a transcriptionalcontrol element refers to the physical and functional relationshipbetween the DNA and promoter such that the transcription of such DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes, binds to and transcribes the DNA.

One or more vectors can be introduced into a cell (e.g., a host cell) toproduce transcripts, proteins, peptides including fusion proteins andpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).In some aspects, the vector is a viral vector. Examples of vectorsinclude, but are not limited to lentiviruses, adenoviral, andadeno-associated viruses. The type of vector can also be selected fortargeting a specific cell type.

The vectors disclosed herein can comprise one or more promoters orregulatory elements or the like. In an aspect, a vector comprises one ormore pol promoters, one or more pol promoters II, one or more poll IIIpromoters, or combinations thereof. Examples of pol II promotersinclude, but are not limited to the retroviral Rous sarcoma virus (RSV)LTR promoter (optionally with the RSV enhancer), the cytomegalovirus(CMV) promoter (optionally with the CMV enhancer), the SV40 promoter,the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. In some aspects,pol II promoters can be engineered to confer tissue specific andinducible regulation of gRNAs. Examples of pol III promoters include,but are not limited to, U6 and H1 promoters. In an aspect, the promoteris U6. In an aspect, the promoter operably linked to the gRNA is a PolIII promoter, human u6, mouse U6, H1, or 7SK.

In some aspects, the compositions described herein (e.g., CRISPR-Cassystems, vectors) can comprise one or more promoters or regulatoryelements. In the instance of two or more promoters or regulatoryelements, said promoters or regulatory elements can be referred to as afirst promoter, a second promoter and so on.

The vector or vector systems disclosed herein can comprise one or morevectors. Vectors can be designed for expression of CRISPR transcripts(e.g., nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. CRISPR transcripts, for example, can be expressed inbacterial cells (e.g., Escherichia coli), insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example, using T7 promoter regulatory sequencesand T7 polymerase.

Generating constructs for the CRISPR/Cas9 system described herein can bea singleplex or multiplexed. The targets of the CRISPR/Cas9 systemdescribed herein can be multiplexed. In some aspects, the vectors can besingleplex vector or multiplex vectors. In some aspects, the singleplexor multiplex vectors can be repression or downregulation vectors orupregulation vectors or a combination thereof.

Vectors can be introduced in a prokaryote, amplified and then theamplified vector can be introduced into a eukaryotic cell. The vectorcan also be introduced in a prokaryote, amplified and serve as anintermediate vector to produce a vector that can be introduced into aeukaryotic cell (e.g., amplifying a plasmid as part of a viral vectorpackaging system). A prokaryote can be used to amplify copies of avector and express one or more nucleic acids to provide a source of oneor more proteins for delivery to a host cell or host organism.Expression of proteins in prokaryotes is often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Vectors can also be a yeast expression vector (e.g.,Saccharomyces cerivisaie).

In some aspects, the vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include but are not limited topCDM8 and pMT2PC. In mammalian cells, regulatory elements control theexpression of the vector. Examples of promoters are those derived frompolyoma, adenovirus 2, cytomegalovirus, simian virus 40, and othersdisclosed herein and known in the art.

In some aspects, the regulatory element is operably linked to one ormore elements of a CRISPR system to drive expression of the one or moreelements of the CRISPR system. CRISPRs are a family of DNA loci that aregenerally specific to a particular species (e.g., bacterial species).The CRISPR locus comprises a distinct class of interspersed shortsequence repeats (SSRs) that were identified in E. coli, and associatedgenes. The repeats can be short and occur in clusters that are regularlyspaced by unique intervening sequences with a constant length.

As used herein, “CRISPR system” and “CRISPR-Cas system” refers totranscripts and other elements involved in the expression of ordirecting the activity of CRISPR-associated (“Cas”) genes, includingsequences encoding a Cas gene, a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system; e.g. guide RNAor gRNA), or other sequences and transcripts from a CRISPR locus. Insome aspects, one or more elements of a CRISPR system is derived from atype I, type II, or type III CRISPR system. In some aspects, one or moreelements of a CRISPR system are derived from a particular organismcomprising an endogenous CRISPR system, such as Streptococcus pyogenes.Generally, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a proto spacer in the context of an endogenous CRISPRsystem).

As used herein, the term “target sequence” refers to a sequence to whicha guide sequence (e.g. gRNA) is designed to have complementarity, wherehybridization between a target sequence and a guide sequence promotesthe formation of a CRISPR complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a CRISPR complex. A targetsequence can comprise any polynucleotide, such as DNA or RNApolynucleotides. In some aspects, a target sequence is located in thenucleus or cytoplasm of a cell. In some aspects, the target sequence canbe within an organelle of a eukaryotic cell (e.g., mitochondrion). Asequence or template that can be used for recombination into thetargeted locus comprising the target sequences is referred to as an“editing template” or “editing polynucleotide” or “editing sequence.”Disclosed herein are target sequences. In an aspect, the targetsequence(s) can be is selected from one or more of the sequences listedin Table 1 and 3.

A guide sequence (e.g. gRNA) can be any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR-Cas system or CRISPR complex to the target sequence. In someaspects, the degree of complementarity between a guide sequence (e.g.gRNA) and its corresponding target sequence is about or more than about50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In some aspects, aguide sequence is about more than about 5, 10, 15, 20, 25, 30, 35, 40,45, 50 or more nucleotides in length or any number in between.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). It is believed that the targetsequence should be associated with a PAM (protospacer adjacent motif);that is, a short sequence recognized the CRISPR complex. The precisesequence and length requirements for the PAM differ depending on theCRISPR enzyme used, but PAMs are typically 2-5 base pair sequencesadjacent the protospacer (that is, the target sequence). A skilledperson will be able to identify further PAM sequences for use with agiven CRISPR enzyme. In an aspect, the PAM comprises NGG (where N is anynucleotide, (G)uanine, (G)uanine). The target sequence corresponds toone or more receptors. In an aspect, the target sequence is a tumornecrosis factor receptor (e.g., TNFR2), interleukin 1 receptor (e.g,IL1R2, IL6R), A-kinase anchor protein 5 (e.g., AKAP5,), a glycoprotein(e.g., gp130) and transient receptor potential cation channel subfamilyV member 1 (TRPV1). In an aspect, the target sequence can be selectedfrom one or more of the sequences listed in Table 1 and Table 3.

Disclosed herein, are gRNA sequences. The disclosed gRNA sequences canbe specific for one or more desired target sequences. In some aspects,the gRNA sequence hybridizes with a target sequence of a DNA molecule orlocus in a cell. In an aspect, the gRNA sequence hybridizes to one ormore target or targets sequences corresponding to including but notlimited to dorsal root ganglion (e.g., peripheral cells), nocicipetiveneurons (CGRP+ (heat sensor); substance P; TRPV1, AKAP 79, AKAP 150 andnerve growth factor (NGF)). In some aspects, the cell can be aeukaryotic cell. In some aspects, the target sequences can be selectedfrom one or more of the sequences listed in Table 1 and Table 3. In someaspects, the cell is a mammalian or human cell. In some aspects, thecell is a mesenchymal stem cell. In some aspects, the cell is an annulusfibrosus cell or nucleus pulposus cell. In some aspects, the cell can beany cell that can be delivered therapeutically to the disc, includingbut not limited to stem cells, or primary cells, such annulus fibrosus(AF) cells, nucleus pulposus (NP) cells, chondrocytes, fibroblasts. Fordirect gene therapy and delivery to the disc, the cell type can be anycell type in the IVD, including but not limited to AF and NP cells, aswell as the chondrocytes in the endplate; and invading immune cells inthe disc, including macrophages, T-cells, neutrophils. In an aspect,gRNA sequences target one or more cell type in the IVD.

In some aspects, the gRNA targets and hybridizes with the targetsequence and directs the RNA-directed nuclease to the DNA locus. In someaspects, the CRISPR-Cas system and vectors disclosed herein comprise oneor more gRNA sequences. In some aspects, the gRNA sequences are listedin Tables 2 and 4. In some aspects, the target sequences can be selectedfrom one or more of the sequences listed in Tables 1 and 3. In someaspects, the CRISPR-Cas system and vectors disclosed herein comprise 2,3, 4 or more gRNA sequences. In some aspects, the CRISPR-Cas systemand/or vector described herein comprises 4 gRNA sequences in a singlesystem. In some aspects, the gRNA sequences disclosed herein can be usedturn one or more genes on (p300core) or off (KRAB).

The compositions described herein can include a nucleic acid encoding aRNA-directed nuclease. The RNA-directed nuclease can be aCRISPR-associated endonuclease. In some aspects, the RNA-directednuclease is a Cas9 nuclease or protein. In some aspects, the Cas9nuclease or protein can have a sequence identical to the wild-typeStreptococcus pyrogenes sequence. In some aspects, the Cas9 nuclease orprotein can be a sequence for other species including, for example,other Streptococcus species, such as thermophdus; Psuedomona aeruginosa,Escherichia coli, or other sequenced bacteria genomes and archaea, orother prokaryotic microogranisms. In some aspects, the wild-typeStreptococcus pyrogenes sequence can be modified. In some aspects, thenucleic acid sequence can be codon optimized for efficient expression ineukaryotic cells.

Disclosed herein, are CRISPR-Cas systems, referred to as CRISPRi (CRISPRinterference), that utilizes a nuclease-dead version of Cas9 (dCas9). Insome aspects, the dCas9 is used to repress expression of one or moretarget sequences (e.g., tumor necrosis factor receptor (e.g., TNFR2),interleukin 1 receptor (e.g, IL1R2, IL6R), A-kinase anchor protein 5(e.g., AKAP5, a glycoprotein (e.g., gp130) and transient receptorpotential cation channel subfamily V member 1 (TRPV1)). In some aspects,the target sequences can be selected from one or more of the sequenceslisted in Table 1 and Table 3. Instead of inducing cleavage, dCas9remains bound tightly to the DNA sequence, and when targeted inside anactively transcribed gene, inhibition of, for example, pol IIprogression through a steric hindrance mechanism can lead to efficienttranscriptional repression.

In some aspects, the CRISPR system can be used in which the nucleus hasbeen deactivated. Further, a KRAB or p300 core can be attached. In someaspects, the KRAB is attached to downregulate one or more genes in acell. In some aspects, the p300core is attached to upregulate one ormore genes in a cell.

In some aspects, the CRISP-Cas system described herein can be used toupregulate or downregulate one or more genes in the same cell. In someaspects, the CRISP-Cas system described herein can also be used toupregulate and downregulate more than one gene or a combination thereofin the same cell. In an aspect, the expression of one or more genes (orgene products) can be decreased. In some aspects, the expression of oneor more genes (or gene products) can be increased. In some aspects, theexpression of one or more genes (or gene products) is increased anddecreased.

In some aspects, the vector comprises a regulatory element operablylinked to an enzyme-coding sequence encoding a CRISPR enzyme (e.g., aCas protein). In some aspects, the CRISPR enzyme is Cas9 and can be fromStreptococcus pyogenes, Streptococcus thermophiles, or TreponemaCenticola. In some aspects, the Cas9 is dCas9. In some aspects, the Cas9protein can be codon optimized for expression in the cell.

In some aspects, dCas9 can be used to silence one or more target genes(e.g., TNFR2, IL1R2, IL6R, AKAP5, gp130 and TRPV1). For example, dCas9can be used to silence one or more genes through steric hindrance withor without an attached domain, such as KRAB. dCas9 is the protein thatinteracts with gRNAs to place the desired editing proteins to specificsites. dCas9 can be used to silence (downregulate or turn off one ormore genes). In some aspects, the dCas9 can be attached to KRAB toknockdown, silence or downregulate one or more genes. In some aspects,the dCas9 can be attached to p300 core to turn on or upregulate one ormore genes. Other proteins can be further attached to dCas9 or includedin the CRISPR-Cas system and/or vectors described herein. For example,T2A, a self-cleaving peptide, can be included. T2A allows selectionmarkers (e.g., GFP, fluorescent proteins, antibiotics) to also beattached. The attachment of such markers can be included to permitdetection or selection of cells expressing the CRISPR-Cas system and/orvectors described herein

Multiple gRNAs can be used to control multiple different genessimultaneously (multiplexing gene targeting), as well as to enhance theefficiency of regulating the same gene target.

TABLE 1 Examples of Target Sequences Target SEQ ID Gene Name SequenceNO. TNFR2 gRNA 1 5′-GAAGGCTGGATGCGTGTTTA-3′ 13 TNFR2 gRNA 25′-TCAAGTGATCTTCCCGCCTC-3′ 14 TNFR2 gRNA 3 5′-GTGAGGGTGAGGCACTAATT-3′ 15TNFR2 gRNA 4 5′-CGGGCTTTCGCTTTCAGTCG-3′ 16 TNFR2 gRNA 55′-AGGTATCGGCCCAGCGATGC-3′ 17 TNFR2 gRNA 6 5′-CATGTCCTAAAATCACGAAC-3′ 18TNFR2 gRNA 7 5′-AATTGTGATACTAGCGGTTA-3′ 19 IL1R2 gRNA 15′-AGGATAAAACTAGGGCCCAT-3′ 20 IL1R2 gRNA 2 5′-CAAGGTTTTACGCTCCCATT-3′ 21IL1R2 gRNA 3 5′-GGGAGGTGACACCCAGTTTA-3′ 22 IL6R gRNA 15′-TAAACACCTGACACACGGTC-3′ 23 IL6R gRNA 2 5′-CGGAAGACTCACCACCGTAA-3′ 24IL6R gRNA 3 5′-AGGGCGTATCAGCCACCAGT-3′ 25 IL6R gRNA 45′-CGGCTTTCGTAACCGCACCC-3′ 26 IL6R gRNA 5 5′-AGAGCCGGGCTCCTGCGGAT-3′ 27gp130 gRNA 1 5′-AGGCTCGTTTACGTAAGTCT-3′ 28 gp130 gRNA 25′-GGAATAACGGGGTCATGAAC-3′ 29 gp130 gRNA 3 5′-CAGTGGCCGCCTGTCGACGA-3′ 30gp130 gRNA 4 5′-CGCACGAACCCCTTGGCGCC-3′ 31 AKAP5 gRNA 15′-TCAACAGGATCACGACCTTA-3′ 32 AKAP5 gRNA 2 5′-ATCGTGGTTCATCGCCAAAC-3′ 33AKAP5 gRNA 3 5′-GCTGCATCTCTATGCGGACA-3′ 34 AKAP5 gRNA 45′-TTAGCGTCTCAGAAAACGCG-3′ 35 AKAP5 gRNA 5 5′-AAACTGTGCATAGAATAGCG-3′ 36AKAP5 gRNA 6 5′-AAGATCAACGTAGGGCGTCG-3′ 37 AKAP5 gRNA 75′-CCTTGCTCCGGGTGCGACCG-3′ 38 AKAP5 gRNA 8 5′-GCGCCCGGGCGGAGCACGAT-3′ 39TRPV1 gRNA 1 5′-AATTAGCTGGGCGCAATGGC-3′ 40 TRPV1 gRNA 25′-GAGTAGGGGTTGGCGTCGAG-3′ 41 TRPV1 gRNA 3 5′-GACGCTAGTTTTGACGTCGC-3′ 42TRPV1 gRNA 4 5′-GAGTCGCTGTGGACGCCCTT-3′ 43 TRPV1 gRNA 55′-TGAAGGCGGTTGCTACTCGA-3′ 44 TRPV1 gRNA 6 5′-AAGGCAGCTGCTTGCATCGC-3′ 45

TABLE 2 Examples of Guide RNA Sequences (gRNAs) Target SEQ ID Gene NameSequence NO. TNFR2 gRNA 1 5′-GAAGGCUGGAUGCGUGUUUA-3′ 46 TNFR2 gRNA 25′-UCAAGUGAUCUUCCCGCCUC-3′ 47 TNFR2 gRNA 3 5′-GUGAGGGUGAGGCACUAAUU-3′ 48TNFR2 gRNA 4 5′-CGGGCUUUCGCUUUCAGUCG-3′ 49 TNFR2 gRNA 55′-AGGUAUCGGCCCAGCGAUGC-3′ 50 TNFR2 gRNA 6 5′-CAUGUCCUAAAAUCACGAAC-3′ 51TNFR2 gRNA 7 5′-AAUUGUGAUACUAGCGGUUA-3′ 52 IL1R2 gRNA 15′-AGGAUAAAACUAGGGCCCAU-3′ 53 IL1R2 gRNA 2 5′-CAAGGUUUUACGCUCCCAUU-3′ 54IL1R2 gRNA 3 5′-GGGAGGUGACACCCAGUUUA-3′ 55 IL6R gRNA 15′-UAAACACCUGACACACGGUC-3′ 56 IL6R gRNA 2 5′-CGGAAGACUCACCACCGUAA-3′ 57IL6R gRNA 3 5′-AGGGCGUAUCAGCCACCAGU-3′ 58 IL6R gRNA 45′-CGGCUUUCGUAACCGCACCC-3′ 59 IL6R gRNA 5 5′-AGAGCCGGGCUCCUGCGGAU-3′ 60gp130 gRNA 1 5′-AGGCUCGUUUACGUAAGUCU-3′ 61 gp130 gRNA 25′-GGAAUAACGGGGUCAUGAAC-3′ 62 gp130 gRNA 3 5′-CAGUGGCCGCCUGUCGACGA-3′ 63gp130 gRNA 4 5′-CGCACGAACCCCUUGGCGCC-3′ 64 AKAP5(79) gRNA 15′-UCAACAGGAUCACGACCUUA-3′ 65 AKAP5(79) gRNA 25′-AUCGUGGUUCAUCGCCAAAC-3′ 66 AKAP5(79) gRNA 35′-GCUGCAUCUCUAUGCGGACA-3′ 67 AKAP5(79) gRNA 45′-UUAGCGUCUCAGAAAACGCG-3′ 68 AKAP5(79) gRNA 55′-AAACUGUGCAUAGAAUAGCG-3′ 69 AKAP5(79) gRNA 65′-AAGAUCAACGUAGGGCGUCG-3′ 70 AKAP5(79) gRNA 75′-CCUUGCUCCGGGUGCGACCG-3′ 71 AKAP5(79) gRNA 85′-GCGCCCGGGCGGAGCACGAU-3′ 72 TRPV1 gRNA 1 5′-AAUUAGCUGGGCGCAAUGGC-3′ 73TRPV1 gRNA 2 5′-GAGUAGGGGUUGGCGUCGAG-3′ 74 TRPV1 gRNA 35′-GACGCUAGUUUUGACGUCGC-3′ 75 TRPV1 gRNA 4 5′-GAGUCGCUGUGGACGCCCUU-3′ 76TRPV1 gRNA 5 5′-UGAAGGCGGUUGCUACUCGA-3′ 77 TRPV1 gRNA 65′-AAGGCAGCUGCUUGCAUCGC-3′ 78

Methods

Methods of designing gRNAs. In some aspects, a commercially availabletool, such as the UCSC genome browser (GRCh37/hg19), can be used toselect sequences for the 5-UTR and the promoter region, 1000 base pairsupstream that can be entered into the CRISPR design tool(crispr.mit.edu). The design tool outputs 20 base pair gRNAs that arefollowed on their 3′ end by the PAM sequence NGG, which is specific tothe CRISPR-Cas9 system derived from Streptococcus pyogenes. The designtool can also score the potential gRNA sequences based on the number ofoff-target sties they may have and how many are within genes. The scoreranges from 0-100, with a higher score meaning less off-target siteswithin genes. Guide RNAs described herein, for example, that had a scoreof 75 and above were selected for further study. The selected gRNAs canthen be entered into the BLAT tool of the UCSC genome browser to inspectfor overlap of gRNAs with DNAse hypersensitivity sites to ensureoverlap. Any site that has DNAse hypersensitivity value above 0.01 canbe targeted with a guide if one is available from the list of guidesgenerated as described above. Additionally, any site that shows greaterthan 10 transcription factor binding sites within a region, asdetermined from ChiP-seq, can also be considered. Generally, the DNAsehypersensitivity data is consistent with these regions. Using thecriteria described above, gRNAs (e.g., 4-7 gRNAs) that are spaced atleast 100 base pairs apart can be selected for performing targeted generepression and screening. In an aspect, TNFR1, TNFR2, IL1R2, IL6R,IL1R1, AKAP5, gp130, and TRPV1 gRNAs guides can be screening using themethod disclosed herein. In an aspect, gRNA sequences from the promoterregion and 5′UTR (crispr.mit.edu) can be selected. In an aspect, gRNAsequences are 20 bp in length followed by a PAM sequence (e.g., NGG). Inan aspect, gRNA sequences with the least off-target sequences and thosethat overlap with DNase sensitivity peaks can be selected.

Disclosed herein are methods of modulating expression of a gene in acell. The method can include one or more of the following steps. First,introducing into a cell, a first nucleic acid. The first nucleic acidcan encode a guide RNA comprising a DNA-binding domain. The nucleic acidcan be operably linked to a regulatory element. The guide RNA describedherein can be complementary to a target nucleic acid sequence disclosedherein comprising the gene. Next, a second nucleic acid encoding atranscriptional regulator protein or domain that modulates the targetnucleic acid expression can be introduced into the cell. The secondnucleic acid can further include a gRNA-binding domain. The secondnucleic acid can be operably linked to a regulatory element. A thirdnucleic acid encoding a Cas9 (e.g., a deactivated nuclease (dCas9))protein can be introduced. In an aspect, nuclease function can beremoved. The third nucleic acid can be operably linked to a regulatoryelement. The Cas9 protein (e.g., dCas9) can interact with the guide RNA,and can be fused to the transcriptional regulator protein. The cell canthen produce the guide RNA. The guide RNA can bind to the dCas9 proteinand the transcriptional regulator protein or domain fused to theDNA-binding domain, and direct the complex (e.g., gRNA/dCas9 complex;the combined product of the gRNA and dCas9 interacting) to the DNAregulatory element encoded in the DNA-binding domain. The guide RNA andthe dCas9 protein, for example, can co-localize to the target nucleicacid sequence. The transcriptional regulator protein or domain canmodulate (increase or decrease) expression of the gene. The gRNAsequence can selected from the group listed in Table 2 and Table 4.

Disclosed herein, are methods for introducing into a cell a CRISPR-Cassystem. In some aspects, the CRISPR-Cas system can include one or morevectors described herein. In some aspects, the method can include one ormore vectors. For example, the vector can comprise a promoter operablylinked to one or more nucleotide sequences encoding a CRISPR-Cas systemgRNA. In some aspects, the gRNA can hybridize with a target sequence ofa DNA molecule in a cell. In some aspects, the vector can also include aregulatory element operably linked to a nucleotide sequence encoding aRNA-directed nuclease. In some aspects, the promoter operably linked toone or more nucleotide sequences encoding a CRISPR-Cas system gRNA andthe regulatory element operably linked to a nucleotide sequence encodinga RNA-directed nuclease can be located on the same or different vectorsof the same system. In some aspects, the method also includes a stepwherein the gRNA targets and hybridizes with the target sequence anddirects the RNA-directed nuclease to the DNA molecule. In some aspects,the gRNA sequence can be selected from the group listed in Table 2 andTable 4. In some aspects, the target sequence can be selected from oneor more of the sequences listed in Table 3 and 4.

Disclosed herein, are methods for introducing into a cell a vector. Insome aspects, the vector can include a promoter operably linked to oneor more nucleotide sequences encoding a CRISPR-Cas system gRNA. In someaspects, the vector can also include a regulatory element operablylinked to a nucleotide sequence encoding a RNA-directed nuclease. Insome aspects, the gRNA sequence can be selected from the group listed inTable 2 and Table 4.

Disclosed herein, are methods for inducing site-specific DNA cleavage ina cell. The method can contacting a cell with a guide RNA. The guide RNAcan be selected from the group listed in Table 2 and Table 2. The guideRNA can include a sequence capable of binding to a target DNA. Themethod can further comprise the following step: contacting the cell witha Cas9 protein. In an aspect, the DNA is in a cell. In an aspect, thecell is a eukaryotic cell. In an aspect, the cell is in an individual.In an aspect, the individual is a human.

The method steps described herein can be carried out simultaneously orsequentially in any order. In some aspects, the DNA can be in a cell. Insome aspects, the cell can be a eukaryotic cell. In some aspects, thecell can be in an individual. In some aspects, the individual can be ahuman.

Method of Treatment

The methods disclosed herein can be useful for the treatment of asubject having lower back pain. The methods disclosed herein can beeffective for targeting one or more receptors, including tumor necrosisfactor receptor (e.g., TNFR2, TNFR1), interleukin 1 receptor (e.g,IL1R2, IL1R1, IL6R), A-kinase anchor protein 5 (e.g., AKAP5,), aglycoprotein (e.g., gp130) and transient receptor potential cationchannel subfamily V member 1 (TRPV1). In some aspects, the method scanalso include the step of administering a therapeutic effective amount ofthe compositions disclosed herein (e.g., a CRISPR-Cas system comprisingone or more vectors comprising: a) a promoter operably linked to one ormore nucleotide sequences encoding a CRISPR-Cas system guide RNA (gRNA),wherein the gRNA hybridizes with a target sequence of a DNA locus in acell; and b) a regulatory element operably linked to a nucleotidesequence encoding a RNA-directed nuclease, wherein components a) and b)are located on the same or different vectors of the same system, whereinthe gRNA targets and hybridizes with the target sequence and directs theRNA-directed nuclease to the DNA locus; wherein the gRNA sequence isselected from the group listed in Table 2 and Table 4. In some aspects,the target sequences can be selected from one or more of the sequenceslisted in Table 1 and Table 3. In some aspects, the methods can furtherinclude the step of identifying a subject (e.g., a human patient) whohas low back pain and then providing to the subject a compositioncomprising the CRISPR-Cas system or vector disclosed herein. In someaspects, the back pain can be caused by degenerative disc disease,dicogenic, one or more facet joints, one or more muscles, inflammationor changes inflammatory cytokines. In some aspects, the subject can beidentified using standard clinical tests known to those skilled in theart. Examples of tests for diagnosing degenerative disc disease includeimaging (e.g., MM, T2 weighted, T1rho). Subjects can also be identifiedas having axial back pain through self-reporting and completing theOswestry low back disability questionnaire.

The therapeutically effective amount can be the amount of thecomposition administered to a subject that leads to a full resolution ofthe symptoms of the condition, disease or pain, a reduction in theseverity of the symptoms of the condition, disease or pain, or a slowingof the progression of symptoms of the condition, disease or pain. Themethods described herein can also include a monitoring step to optimizedosing. The methods can also include the step of determining the nucleicacid sequence of the specific cytokine or receptor present in asubject's DNA and then design the CRISPR-Cas system or vectors tocomprise specific DNA binding domain sequences or gRNA sequences. Thecompositions described herein can be administered as a preventivetreatment or to delay or slow the progression of degenerative changes orat the time of surgery.

The compositions disclosed herein can be used in a variety of ways. Forinstance, the compositions disclosed herein can be used for directdelivery of modified therapeutic cells, or lentivirus. The compositionsdisclosed herein can be used or delivered or administered at any timeduring the treatment process (e.g., during an already conducted surgery,when either a spinal fusion is being conducted, or a microdiscectomy).The compositions described herein including cells or a virus can bedelivered to the affected level in the microdiscetomy case, or to theadjacent levels to stop adjacent segment disease in the spinal fusioncase.

In some aspects, the compositions disclosed herein can be administeredor delivered to peripheral neurons (e.g., to regulate inflammatoryinteractions with the pain sensing neurons). Such treatment can becarried out if this type of interaction is suspected in back pain.

The dosage to be administered depends on many factors including, forexample, the route of administration, the formulation, the severity ofthe patient's condition/disease/pain, previous treatments, the patient'ssize, weight, surface area, age, and gender, other drugs beingadministered, and the overall general health of the patient includingthe presence or absence of other diseases, disorders or illnesses.Dosage levels can be adjusted using standard empirical methods foroptimization known by one skilled in the art. Administrations of thecompositions described herein can be single or multiple (e.g., 2- or 3-,4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Further,encapsulation of the compositions in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) can improve theefficiency of delivery.

The therapeutically effective amount of the compositions describedherein can include a single treatment or a series of treatments (i.e.,multiple treatments or administered multiple times). Treatment durationusing any of compositions disclosed herein can be any length of time,such as, for example, one day to as long as the life span of the subject(e.g., many years). For instance, the composition can be administereddaily, weekly, monthly, yearly for a period of 5 years, ten years, orlonger. The frequency of treatment can vary. For example, thecompositions described herein can be administered once (or twice, threetimes, etc.) daily, weekly, monthly, or yearly for a 15 period of 5years, ten years, or longer.

In some aspects, the compositions disclosed herein can also beco-administered with another therapeutic agent or in combination withmicrodiscectomy or spinal fusion surgery.

In some aspects, the methods disclosed herein also include treating asubject having degenerative disc disease. In some aspects, the methodsdisclosed herein can include the step of determining TNF, IL1, IL6, IL8,IFN-gamma and/or IL17 levels in a subject. In some aspects, thedisclosed methods can further include the step of administering to thesubject a pharmaceutical composition comprising a nucleic acid sequenceencoding a CRISPR-associated endonuclease (e.g., deactivatedendonuclease) and one or more guide RNAs, wherein the guide RNA isselected from the group listed in Table 2 and Table 4. In some aspects,the CRISPR-associated endonuclease is optimized for expression in ahuman cell.

Pharmaceutical Compositions

As disclosed herein, are pharmaceutical compositions, comprising thecompositions disclosed herein. For example, disclosed are pharmaceuticalcompositions, comprising a vector or CRISPR-Cas system comprising one ormore vectors comprising: a) a promoter operably linked to one or morenucleotide sequences encoding a CRISPR-Cas system guide RNA (gRNA),wherein the gRNA hybridizes with a target sequence of a DNA locus in acell; and b) a regulatory element operably linked to a nucleotidesequence encoding a RNA-directed nuclease, wherein components a) and b)are located on the same or different vectors of the same system, whereinthe gRNA targets and hybridizes with the target sequence and directs theRNA-directed nuclease to the DNA locus; wherein the gRNA sequence isselected from the group listed in Table 2 and Table 4. In some aspects,the target sequence can be selected from one or more of the sequenceslisted in Table 1 and Table 3. In some aspects, the pharmaceuticalcompositions comprise the any one of the CRISPR-Cas system disclosedherein. In some aspects, the pharmaceutical composition comprises thenucleic acid sequence of any of the vectors or CRISPR-Cas systemsdisclosed herein. In some aspects, the pharmaceutical compositionsfurther comprise a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers tosolvents, dispersion media, coatings, antibacterial, isotonic andabsorption delaying agents, buffers, excipients, binders, lubricants,gels, surfactants that can be used as media for a pharmaceuticallyacceptable substance. The pharmaceutically acceptable carriers can belipid-based or a polymer-based colloid. Examples of colloids includeliposomes, hydrogels, microparticles, nanoparticles and micelles. Thecompositions can be formulated for administration by any of a variety ofroutes of administration, and can include one or more physiologicallyacceptable excipients, which can vary depending on the route ofadministration. Any of the nucleic acids and vectors and gRNAs describedherein can be administered in the form of a pharmaceutical composition.

As used herein, the term “excipient” means any compound or substance,including those that can also be referred to as “carriers” or“diluents.” Preparing pharmaceutical and physiologically acceptablecompositions is considered routine in the art, and thus, one of ordinaryskill in the art can consult numerous authorities for guidance ifneeded. The compositions can also include additional agents (e.g.,preservatives).

The pharmaceutical compositions as disclosed herein can be prepared fororal or parenteral administration. Pharmaceutical compositions preparedfor parenteral administration include those prepared for intravenous (orintra-arterial), intramuscular, intervertebral subcutaneous, facetjoint, dorsal root ganglion, intrathecal or intraperitonealadministration. Paternal administration can be in the form of a singlebolus dose, or may be, for example, by a continuous pump. In someaspects, the compositions can be prepared for parenteral administrationthat includes dissolving or suspending the CRISPR-Cas systems, nucleicacids, polypeptide sequences or vectors in an acceptable carrier,including but not limited to an aqueous carrier, such as water, bufferedwater, saline, buffered saline (e.g., PBS), and the like. One or more ofthe excipients included can help approximate physiological conditions,such as pH adjusting and buffering agents, tonicity adjusting agents,wetting agents, detergents, and the like. Where the compositions includea solid component (as they may for oral administration), one or more ofthe excipients can act as a binder or filler (e.g., for the formulationof a tablet, a capsule, and the like). Where the compositions areformulated for application to the skin or to a mucosal surface, one ormore of the excipients can be a solvent or emulsifier for theformulation of a cream, an ointment, and the like.

In some aspects, the CRISPR-Cas system disclosed herein can be directlyinjected via a lentivirus into the IVD without a carrier or with abiomaterial carrier. Any hydrogel or biomaterial designed for viralvector delivery can be used.

In some aspects, the cells can be administered to a desired locationwith or without a biomaterial carrier.

In some aspects, a virus comprising one or more vectors disclosed hereinor the CRISPR-Cas system disclosed herein can be administered with orwithout a carrier to one or more peripheral nerves. In some aspects, theone or more peripheral nerves can be innervating the disc. Thus, theadministration of the virus comprising any one of the compositionsdisclosed herein can be delivered (e.g., injected) to the disc site ordorsal root ganglion (DRG).

In some aspects, the compositions disclosed herein are formulated forintervertebral administration

The pharmaceutical compositions can be sterile and sterilized byconventional sterilization techniques or sterile filtered. Aqueoussolutions can be packaged for use as is, or lyophilized, the lyophilizedpreparation, which is encompassed by the present disclosure, can becombined with a sterile aqueous carrier prior to administration. The pHof the pharmaceutical compositions typically will be between 3 and 11(e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7and 8). The resulting compositions in solid form can be packaged inmultiple single dose units, each containing a fixed amount of theabove-mentioned agent or agents, such as in a sealed package of tabletsor capsules. The composition in solid form can also be packaged in acontainer for a flexible quantity, such as in a squeezable tube designedfor a topically applicable cream or ointment. The compositions can alsobe formulated as powders, elixirs, suspensions, emulsions, solutions,syrups, aerosols, lotions, creams, ointments, gels, suppositories,sterile injectable solutions and sterile packaged powders. The activeingredient can be nucleic acids or vectors described herein incombination with one or more pharmaceutically acceptable carriers. Asused herein “pharmaceutically acceptable” means molecules andcompositions that do not produce or lead to an untoward reaction (i.e.,adverse, negative or allergic reaction) when administered to a subjectas intended (i.e., as appropriate).

In some aspects, the CRISPR-Cas system, vectors, gRNAs and nucleic acidsequences as disclosed herein can be delivered to a cell of the subject.In some aspects, such action can be achieved, for example, by usingpolymeric, biodegradable microparticle or microcapsule delivery vehicle,sized to optimize phagocytosis by phagocytic cells (e.g., macrophages).

In some aspects, the formulations include any that are suitable for thedelivery of a virus (e.g., lentivirus) and cells. In an aspect, theroute of administration includes but is not limited to injection intothe disc, adjacent to the disc, or directly to the DRG for peripheralneuron transduction. Such administration can be done without surgery, orwith surgery.

Kits

The kits described herein can include any combination of thecompositions (e.g., CRISR-Cas system or vectors) described above andsuitable instructions (e.g., written and/or provided as audio-, visual-,or audiovisual material). In an aspect, the kit comprises apredetermined amount of a composition comprising any one of theCRISPR-Cas or vectors disclosed herein. The kit can further comprise oneor more of the following: instructions, sterile fluid, syringes, asterile container, delivery devices, and buffers or other controlreagents.

EXAMPLES Example 1: CRISPR Epigenomic Editing of AKAP 150 in DRG NeuronsAbolished Degenerative IVD Induced Sensitization of Sensory Neurons

Materials and Methods

Experimental Overview.

A set of four experiments was conducted in the degenerative IVDenvironment (e.g., inflammatory cytokines and low pH) on thesensitization of DRG neurons to noxious stimuli and to elucidate thefactors and mechanisms mediating this sensitization. In the DegenerativeIVD Conditioned Media Exposure experiments, rat DRG neurons weresubjected to a range of temperatures during human degenerative andhealthy IVD conditioned media exposure to determine the ability offactors released from degenerative IVD tissue to sensitize neurons andthe magnitude of that sensitization. Following the degenerative IVDconditioned media exposure experiment, the IL-6 Blocking Experimentswere performed to determine the role of IL-6 in the degenerative IVDsensitization of peripheral neurons. Once IL-6 was implicated as amediator of neuron sensitization, the AKAP Inhibition Experiments wereconducted to determine the mechanism by which IL-6 sensitizes DRGneurons. Following TRPV1 involvement in DRG sensitization, the AcidicpH-Degenerative IVD Conditioned Media Exposure experiments wereconducted to determine the role of pH in DRG neuron sensitization andits ability to interact with the observed inflammation drivensensitization.

Once the IL-6/AKAP/TRPV1 pathway was identified as the main pathway fordegenerative IVD induced neuron sensitization, the potential of CRISPRepigenome editing of neurons to regulate degenerative IVD inducedsensitization via the targeting of AKAP in this pathway wasinvestigated. Lentiviral constructs carrying the CRISPR epigenomeediting system that targeted the AKAP gene promoter were designed,built, and validated for their ability to regulate AKAP expression inperipheral neurons. Validated CRISPR AKAP targeting epigenome editingvectors were selected, and delivered to DRG neurons prior to exposure todegenerative IVD sensitization model and tested for an ability toregulate previously demonstrated sensitization via IL-6/AKAP/TRPV1pathway.

Degenerative Intervertebral Disc (IVD) Conditioned Media.

IVD tissue was obtained from five patients undergoing surgicalintervention for axial back pain, degenerative disc disease, and lumbarspondylosis. IVD tissue was extracted from lumbar discs for allpatients. Patients showed signs of degenerative disc disease on magneticresonance imaging (MRI) and reported axial back pain. Additionally,non-degenerative IVD tissue was obtained from three trauma patients withno prior history of axial back or neck pain. IVD tissue was transferredinto a glass petri dish, washed twice in washing medium (DMEM-HG (LifeTechnologies) supplemented with 1% gentamycin (Gibco), 1% kanamycin(Sigma) and 1% fungizone (Gibco)) and cut into small pieces (˜3 mm²).Next, the IVD tissue was weighed, transferred to a 75 cm² tissue cultureflask, and cultured in DMEM-HG supplemented with 50 g/ml ascorbic acid(Life Technologies), 5 μg/ml gentamicin, and 0.125 μg/ml fungizone at amedia to tissue ratio of 3.5 ml/g for 48 hours (37° C. and 5% CO₂)[33].After incubation, IVD conditioned media was collected, and stored at−80° C. until needed.

Dorsal Root Ganglion (DRG) Neuron Cell Culture.

Postnatal (p 1-p4) Sprague Dawley rat DRGs were explanted, placed inL-15 medium (Gibco), and cleaned of anterior and posterior roots andconnective tissue. Ganglia were dissociated in 2 mL of L-15 mediumsupplemented with 500 μL of collagenase IV (1.33%, WorthingtonBiochemical) for 30 minutes at 37° C. The cell suspension wascentrifuged at 1000 RPM for 5 minutes after which the supernatant wasaspirated and the cells were incubated in 4 mL of DMEM/F-12 supplementedwith 1 mL of 1% trypsin (Worthington Biochemical) plus 50 μL of 1% DNaseI (Worthington Biochemical) for 20 minutes at 37° C. Followingincubation, 1 mL of soybean trypsin inhibitor (SBTI) (WorthingtonBiochemical) was added to the cell suspension and the cell suspensionwas centrifuged at 1000 RPM for 5 minutes. Next, the supernatant wasaspirated, cells were resuspended in 1 mL of DMEM/F-12 plus 50 μL of 1%DNase and triturated through a fire polished Pasteur pipette. Rat DRGneurons were seeded onto laminin (Life Technologies) coated 35 mm tissueculture dishes at a density of 50,000 cells per dish and cultured in 1.5mL of SATO⁻ medium (DMEM/F12 supplemented with 2.2% SATO-mix, 1%transferrin (Sigma), 2% insulin (Sigma), 1% Glutamax (Invitrogen), 0.5%gentamicin, and 2.5 S NGF (long/ml, Worthington Biochemical) for 2-6days until experiment.

Calcium Imaging of Action Potentials.

Rat DRG neurons were loaded with the calcium indicator dye Fluo-4AM(Molecular Probes, 3 μM) and incubated in the dark at 37° C. for 1 hour.Fluorescent measurements of calcium were performed using a multi-photonmicroscope (Bruker/Prairie View, excitation 810 nm, emission 545 nm, 0.5Hz). Neurons were incubated at 37° C. for 15 minutes to establish thebaseline calcium signal and then exposed to heat stimuli for two minuteswhile imaging. Cells were returned to the baseline temperature for 5minutes between exposures to elevated temperatures. 100 DRG neurons wereimaged and analyzed for each treatment group.

Image analysis was conducted using Fiji software[50]. The backgroundsignal was subtracted from each cell and the mean baseline (F₀(t)) wascalculated at 37° C.

$\begin{matrix}{\frac{\Delta \; F}{F} = \frac{\left( {{F(t)} - {F_{0}(t)}} \right)}{F_{0}(t)}} & \lbrack 1\rbrack\end{matrix}$

was calculated for each cell across the entire experiment. The baselinemean and standard deviation of ΔF/F was calculated for all cells at 37°C. Neurons were considered to be firing (generating an action potential)in response to heat stimuli if the AF/F for the cell was 3 standarddeviations greater than the mean baseline value[19] at 37° C. (FIG. 1)in response to heat stimulation.

Degenerative IVD Conditioned Media Exposure.

Media were replaced with fresh SATO-media and 0.75 ml of unconditionedDMEM (control media, n=5), degenerative IVD conditioned (n=5 patients),or healthy IVD conditioned media (n=3 patients) and cultured for 24hours (37° C., 5% CO₂). Following incubation, neurons were loaded withthe calcium indicating dye Fluo-4AM as described in the Calcium Imagingof Action Potentials section. Neurons were incubated at 37° C. for 15minutes to establish a baseline calcium signal and then exposed to heatof 38, 39, 40, 42, and 44° C. for two minutes while imaging. Cells werereturned to the baseline temperature for 5 minutes between exposures toelevated temperatures.

IL-6 Blocking Experiments.

Mouse monoclonal anti-human IL-6 antibody (Life Technologies, 20 μg/mL)or mouse isotype control antibody (Life Technologies, 20 μg/mL) wereadded to degenerative IVD conditioned media and incubated at 37° C. forthree hours prior to addition to neurons as described in theDegenerative IVD Conditioned Media Exposure section (n=5 patients).Degenerative IVD conditioned media not receiving antibody treatment andcontrol media (DMEM supplemented with 5 μg/ml gentamicin and 0.125 μg/mlfungizone) were incubated at 37° C. for three hours prior to addition toneurons as described in the Degenerative IVD Conditioned Media Exposuresection (n=5). Neurons were incubated at 37° C. for 15 minutes toestablish a baseline calcium signal and then exposed to heat stimulationof 38, 39, 40, 42, and 44° C. for two minutes while imaging. Cells werereturned to the baseline temperature for 5 minutes between exposures toelevated temperatures.

AKAP Inhibition Experiments.

DRG neurons were loaded with the calcium dye Fluo-4AM (3 μM) andincubated in the dark at 37° C. for 1 hour. The AKAP inhibitor peptideSt-Ht31 (Promega, 50 μM) or the control peptide St-Ht31P (Promega, 50μM) were added to the DRG neurons and incubated at 37° C. for 15minutes. Following incubation, degenerative IVD conditioned media (n=3patients) or control media were added to the peptide exposed neurons andincubated for 15 minutes (n=3). Additionally, separate groups of neuronswere exposed to control media or degenerative IVD conditioned mediawithout peptide exposure under the same conditions (n=3 patients).Following this incubation, a 15 minute baseline calcium signal at 37° C.was established and cells were exposed to heat stimulation of 39° C.while imaging.

Acidic pH-Degenerative IVD Conditioned Media Exposure.

Experiments were conducted as described in the Degenerative IVDConditioned Media Exposure section (n=4 patients) with the followingexperimental treatment groups: normal pH (7.4) control media (DMEM),normal pH (7.4) IVD conditioned media, low pH (6.5) control media, andlow pH (6.5) IVD conditioned media. Control media (DMEM) or degenerativeIVD conditioned media were equilibrated in an incubator for 24 hours(37° C., 5% CO₂). Following incubation, the pH of the acidic pH groupswas lowered to a pH of 6.5 by the addition of HCL (1M, Sigma-Aldrich)and the normal pH groups maintained at a pH of 7.4.

Lentiviral CRISPR Epigenome Editing Vector Construction.

CRISPR epigenome editing vectors were created that co-expressesdCAS-KRAB-T2A-GFP and gRNAs that target AKAP 150. First, the promoterregion of rat AKAP 150 are screened for gRNA target sequences with thenecessary adjacent protospacer adjacent motif (PAM: -NGG) and selectedbased on minimizing off-target binding sites using a publicallyavailable algorithm[21]. Four guides are screened per promoter regionthat target every ˜250 bp (data and sequences obtained from the UCSCgenome browser[27]). The non-target guide oligonucleotide was designedas a scramble DNA sequence that does not match the rat genome.Oligonucleotides are obtained (University of Utah DNA/Peptide SynthesisCore), hybridized, phosphorylated and cloned into gRNA expressingplasmids (addgene plasmid 47108) using BbsI sites. To produce alentiviral vector that co-expresses dCAS-KRAB-T2A-GFP and gRNA, gRNAcassette are cloned via PCR and inserted into 3^(rd) generationlentiviral transfer vector that expresses dCas-KRAB-T2A-GFP under thecontrol of the human UbC promoter via BsmBI sites. To producetransduction media (DMEM), epigenome editing vector constructs wereproduced in HEK 293T cells using a previously reported method[49] andstored at −80° C. until use. For transduction of rat DRG neurons, mediawere removed and replaced with transduction media supplemented withpolybrene (8 μg/ml) and cells were cultured for 24 hours. The next day,viral media was removed replaced with fresh SATO-medium. Transduced DRGneurons were cultured in SATO-medium under standard cell cultureconditions until experiments were performed.

AKAP150 Quantitative Reverse-Transcription PCR.

Four days following transduction, cells were harvested for total RNAusing the PureLink RNA microscale kit (LifeTech). cDNA synthesis wasconducted using the High Capacity cDNA RT kit (Lifetech). qRT-PCR usingTaqman Universal PCR Master Mix(LifeTech) was performed with the TaqManGene Expression Detection Assay (LifeTech) with oligonucleotide primersfor AKAP 150 and GAPDH. Results are expressed as fold increase in mRNAexpression of AKAP 150 normalized to GAPDH expression using the ΔΔ C_(t)method.

AKAP Epigenome Edited Neuron Sensitization.

DRG neurons were transduced with epigenome editing lentiviral constructstargeting the AKAP 150 gene promoter region or a non-target gRNA, asdescribed above, and cultured in SATO-medium for 4 days followingremoval of lentivirus. Successful transduction was verified viafluorescent imaging of GFP in transduced neurons. Following the cultureperiod, transduced and naïve (non-transduced) DRG neurons were exposedto degenerative IVD conditioned media or control media (DMEM) andincubated for 24 hours. Following incubation, neurons were loaded withthe calcium indicator dye rhod-2AM (Molecular Probes, 3 μM) andincubated in the dark at 37° C. for 1 hour. Fluorescent measurements ofcalcium were performed using a multi-photon microscope (Bruker/PrairieView, excitation 1105 nm, emission 585 nm, 0.5 Hz). Neurons wereincubated at 37° C. for 15 minutes to establish a baseline calciumsignal and then exposed to heat stimuli of 38, 39, 40, 42, and 44° C.for two minutes while imaging. Cells were returned to the baselinetemperature for 5 minutes between exposures to elevated temperatures.

Curve Fitting of Neuron Firing.

Data from the conditioned media and IL-6 blocking experiments were fitto the sigmoidal Boltzmann equation:

$\begin{matrix}{y = {\min + \frac{\left( {\max - \min} \right)}{1 + {\exp \left( \frac{T_{50} - x}{slope} \right)}}}} & \lbrack 2\rbrack\end{matrix}$

with the percentage of neurons firing plotted as a function oftemperature. Each trial was individually fit to produce values for T50and max. T50 was defined as the temperature at which 50 percent of themaximum response occurs. The max was defined as the maximum percentageof neurons firing predicted by the curve fitting.

Statistical Analysis.

Degenerative IVD conditioned media exposure and IL-6 blocking experimentdata were analyzed by two-way analysis of variance (ANOVA) on repeatedmeasures with Tukey's post hoc test, treating media condition andtemperature as factors. AKAP inhibitor experiment data, T50 and maximumresponse data from IL-6 blocking experiments were analyzed by one-wayANOVA with Tukey's post hoc test, treating media condition as thefactor. Degenerative IVD pH level experiment data were analyzed bytwo-way ANOVA with Tukey's post hoc test, treating media condition andtemperature as factors. Significance was tested at α=0.05 for allstatistical analyses.

Results

Degenerative IVD Conditioned Media Triggers Sensitization of DRG Neuronsto Heat Stimuli.

The percentage of rat DRG neurons firing when exposed to degenerativeIVD conditioned media was significantly elevated over the neurons firingin the control media group (p<0.05) and healthy IVD conditioned mediagroup (p<0.05) at temperatures as low as 38° C. (FIG. 2A). In addition,the percentage of neurons firing when exposed to healthy IVD conditionedmedia was similar to the percentage of neurons firing in the controlmedia group at all temperatures tested (FIG. 2A).

The percentage of neurons firing as a function of temperature was welldefined by curve fitting the data to the Boltzmann equation for thecontrol media (r²=0.97±0.02), healthy IVD conditioned media(r²=0.97±0.03), and degenerative IVD conditioned media (r²=0.97±0.02)treatment groups (FIG. 2A). The T50, the temperature at which half themaximum firing response occurs, and maximum firing response werecalculated via Boltzmann equation fits to each trial. The T50 value ofneurons exposed to thermal stimuli in the presence of degenerative IVDconditioned media (38.25° C.±0.77) was significantly lower than the T50values in both the control media (39.25° C.±0.13, p<0.05) and thehealthy IVD conditioned media treatment groups (39.12° C.±0.38,p<0.05)(FIG. 2B). In addition, the maximum firing response of neuronsexposed to thermal stimuli in the presence of degenerative IVDconditioned media (73.69%±6.5) was significantly greater than themaximum response in the control media (52.69%±9.42, p<0.05) and healthyIVD conditioned media treatment groups (51.25%±11.58, p<0.05) (FIG. 2C).

IL-6 is the Primary Mediator of Sensitization of DRG Neurons byDegenerative IVD Conditioned Media.

The results from the original sensitization experiments were repeatedand confirmed as the percentage of rat DRG neurons firing when exposedto heat stimuli in the presence of degenerative IVD conditioned mediawas significantly elevated (p<0.05) when compared to the percentage ofneurons firing in the control media group at temperatures as low as 38°C. (FIG. 3A). When rat DRG neurons were exposed to heating in thepresence of degenerative IVD conditioned media supplemented with IL-6blocking antibody, the firing response returned to control media levels(p=1.0) for all temperatures tested (FIG. 3A) and was significantly less(p<0.05) than the percentage of neurons firing in the degenerative IVDconditioned media group at all temperatures above 38° C. (FIG. 3A). Incontrast, addition of the isotype control antibody had no effect onfiring response compared to degenerative IVD conditioned media group(p=0.895) (FIG. 3A). Furthermore, the IL-6 sensitization effect was seenin all patients tested, as all patients showed a decreased (p<0.05)firing response after IL-6 blocking antibody exposure (FIGS. 3A, D).

The number of neurons firing as a function of temperature was welldefined by curve fitting the data to the Boltzmann equation for thecontrol media (r²=0.97±0.02), degenerative IVD conditioned media(r²=0.97±0.02), IL-6 blocking antibody (r²=0.98±0.02), and isotypecontrol antibody (r²=0.94±0.04) treatment groups (FIG. 3A). The T50 andmaximum firing response were calculated via Boltzmann equation fits toeach trial. The T50 value of neurons exposed to heat stimuli in thepresence of conditioned media supplemented with IL-6 blocking antibody(39.53° C.±0.4) returned to control media levels (39.25° C.±0.13,p=0.993) and was significantly greater than the T50 values of neurons inboth the conditioned media (38.25° C.±0.77, p=0.004) and isotype controlantibody groups (38.16° C.±0.4, p=0.003) (FIG. 3B). In addition, themaximum firing response of DRG neurons exposed to heat stimuli in thepresence of conditioned media supplemented with IL-6 blocking antibody(44.6%±11%) returned to control media levels (52.7%±9.4%, p=0.534) andis significantly less than the maximum firing response of neurons in theconditioned media (73.7%±6.5%, p<0.0001) and isotype control antibodygroups (66%±2.8%, p=0.001) (FIG. 3C).

Inhibition of AKAP Abolishes IL-6 Mediated Sensitization of Rat DRGNeurons by Degenerative IVD Conditioned Media.

Sensitization by degenerative IVD conditioned media was once againconfirmed as the percentage of DRG neurons firing at 39° C. exposed todegenerative IVD conditioned media (59.72%±18.9%) was significantlygreater (p=0.007) than the percentage of neurons firing in the controlgroup (14.36%±2.3%, FIG. 4). When neurons were exposed to degenerativeIVD conditioned media in the presence of the AKAP inhibitor St-Ht31, thepercentage of neurons firing returned to baseline levels (15.51%±2.7%)and was not significantly different from the control media group(p=0.998, FIG. 4). In addition, the percentage of neurons firing in thedegenerative IVD conditioned media supplemented with the control peptideSt-Ht31P (61.91%±13.9%) was significantly greater than the control media(p=0.006) and the St-Ht31 groups (p=0.006), but not significantlydifferent from the degenerative IVD conditioned media group (p=0.996,FIG. 4).

FIG. 4 shows the results of the experiments described above as apercentage of rat DRG neurons firing exposed to 39° C. in the presenceof control media, degenerative IVD conditioned media, degenerative IVDconditioned media supplemented with the AKAP inhibitor peptide St-Ht31(50 μM) or degenerative IVD conditioned media supplemented with thecontrol peptide St-Ht31P (50 μM). n=3 for all groups tested.

Degenerative IVD Conditioned Media and Pathological Acidic pH LevelsSynergistically Enhance Sensitization of Rat DRG Neurons to Heat Stimuliand Trigger Spontaneous Neuron Firing.

The percentage of rat DRG neurons firing when exposed to pH 6.5degenerative IVD conditioned media was significantly elevated (p<0.05)over all other media conditions tested at 37° C., 38° C., and 39° C. At37° C., spontaneous neuron firing occurred in neurons in the pH 6.5degenerative IVD conditioned media group (13±4.2%, FIG. 5), which hadnot been observed in any other experiment or group tested.

The T50 values of neurons in the pH 6.5 degenerative IVD conditionedmedia group (37.32±0.39° C., p=0.02, FIG. 5B) was significantlydecreased compared to the degenerative IVD conditioned media(38.48±0.28° C.), the pH 6.5 control media (39.76±0.76° C., p=0.01), andthe control media (39.62±0.24° C., p=0.02) groups (FIG. 5).

FIG. 5 shows the results from the experiments described above as thepercentage of rat DRG neurons firing exposed to heat stimuli in thepresence of control media (pH 7.4 degenerative IVD conditioned media (pH7.4), or pH 6.5 degenerative IVD conditioned media (n=4) *=p<0.05 whencompared to the control media group, #=p<0.05 when comparing pH 6.5degenerative IVD conditioned media group to degenerative IVD conditionedmedia group (pH 7.4).

Epigenome Editing of AKAP Promoter in Rat DRG Neurons AbolishesDegenerative IVD Mediated Sensitization of Rat DRG Neurons.

Transduction of DRG neurons with CRISPR epigenetic editing lentiviralvectors targeting AKAP 150 (FIG. 6C) regulated expression of AKAP 150(FIG. 6D) with AKAP guide 4 exhibiting maximum down-regulation whencompared to DRG neurons transduced with non-target lentiviral vectors(21.03% of non-target expression, p<0.05).

The percentage of neurons firing in naïve (non-transduced) neurons andneurons receiving non-targeting lentiviral vectors subjected to thermalstimuli in the presence of degenerative IVD conditioned media weresignificantly elevated when compared to the control media group (p<0.05)at all temperatures tested. When AKAP 150 epigenome edited neurons wereexposed to degenerative IVD conditioned media, the percentage of neuronsfiring returned to baseline levels (FIG. 6E) and were not significantlydifferent from control media levels. From the curve fitting, the T50value of AKAP egigenomically edited neurons exposed to degenerative IVDconditioned media (38.7±0.29° C.) was significantly lower than T50 valueof naïve (37.94±0.07° C., p=0.003) and non-target epigenomically editedneurons (38.7±0.29° C., p=0.01), yet not significantly different fromthe control media group(38.99±0.39° C., p=0.3298)(FIG. 6F). The maximumresponse of AKAP epigenomically edited neurons exposed to degenerativeIVD conditioned media (50.63±6.59%) returned to control levels(51.12±8.02%, p=0.9959) and was significantly decreased compared to themaximum response of naïve (74.48±11.06%, p=0.001) and non-targetepigenomically edited neurons (69.14±4.93%, p=0.001) exposed todegenerative IVD conditioned media (FIG. 6G).

Discussion

The interactions between the degenerative IVD environment (inflammatorycytokines and pH) and peripheral neurons were modeled and investigatedto provide insight into the underlying mechanisms of discogenic backpain. The data presented herein demonstrates that degenerative IVDproduces specific factors/cytokines capable of directly sensitizing DRGneurons to heat stimuli and that sensitization is enhanced at pH levelsexperienced by neurons in the degenerative IVD. This sensitization ledto spontaneous firing of neurons and a T50 firing threshold (37.32±0.39°C.) near resting core body temperature, but was not observed inunconditioned media or media conditioned with healthy IVD. Blocking IL-6in the conditioned media abolished the entirety of this sensitization,and sensitization was also abolished after inhibiting AKAP 150,indicating sensitization occurs through the IL-6/AKAP/TRPV1 pathway.Once the IL-6/AKAP/TRPV1 pathway was established as mediatingsensitization, the ability of CRISPR epigenome editing of peripheralneurons to regulate sensitization was investigated. This datademonstrates that epigenome editing of AKAP 150 expression in rat DRGneurons abolishes degenerative IVD sensitization of neurons to thermalstimuli while these neurons maintain their non-pathological firingresponse. Together, these results implicate the synergistic effects ofacidic pH and the activation of the IL-6/AKAP/TRPV1 as the underlyingmechanism for degenerative IVD induced neuron sensitization,demonstrates CRISPR epigenomic editing of AKAP expression regulatesneuron sensitization and establishes CRISPR epigenome editing ofnociceptive neurons as a possible treatment strategy for discogenicpain.

Factors released from degenerative IVDs sensitize DRG neurons to noxiousstimuli, particularly heating were demonstrated. Previous studies havedemonstrated increased cytokine levels (e.g. TNF-α, IL-1β, IL-6, andIL-8) in painful IVDs[10,36,37,52,53] and that inflammatory cytokinesare capable of sensitizing rodents to thermal and mechanical stimuli inthe paw in models of radiculopathy and peripheral neuropathy[5,42,45].The findings disclosed herein demonstrate an ability for thedegenerative IVD conditions, but not healthy IVD tissue, to directlylead to afferent neuron sensitization. These findings support thehypothesis that inflammation driven sensitization of nociceptive neuronsin the degenerative IVD could contribute to discogenic back pain viasensitization and establish an in vitro model, which can be used tostudy these interactions and screen novel therapeutics.

In this study, the degenerative IVD conditioned media combined withacidic pH was capable of inducing spontaneous neuron firing andsensitizing afferent neurons to noxious stimuli at temperatures as lowas 37° C. Under these sensitized conditions, the T50 value (37.3° C.)falls within the normal core body temperature range (36.1-37.8° C.) andthe maximum response is observed just above this range (38° C.), with13% of neurons spontaneously firing at the mean core temperature of 37°C. Since the majority of neurons innervating the IVD are nociceptiveneurons that co-express CGRP and TRPV1, a pathological ability for TRPV1channels to fire at sub 38° C. temperatures would provide a mechanismfor nociceptive signaling in the degenerative IVD. This data suggeststhat TRPV1 in the disc may play a role in discogenic back pain due tosynergistic pH and inflammatory cytokine mediated sensitization.

This data indicates that IL-6 is the main mediator of the neuronsensitization observed after exposure to degenerative IVD conditionedmedia. Multiple inflammatory cytokines have been observed in thepathological IVD, which includes TNF-α, IL-1β, IL-6, andIL-8[10,36,37,52,53]. IL-1β and TNF-α[35,36,51] are the most commoncytokines investigated in disc pathology due to their role inextracellular matrix breakdown and implication in rodent models ofradiculopathy. TNF-α, IL-1β and IL-6 have all been hypothesized tocontribute to discogenic back pain; however, this data demonstrates thatblocking IL-6 action in the degenerative conditioned media abolished thethermal sensitivity and demonstrated IL-6 as a main mediator ofinflammatory sensitization of neurons in these experiments and not TNF-αor IL-1β. Furthermore, IL-6 dependent sensitization was observed inneurons exposed to conditioned media from all patients tested suggestingthe same mechanism may exist in the larger patient population. It'simportant to note that DRG neurons do not express membrane bound IL-6receptor but rather IL-6 signals via trans-signaling through a complexformed by IL-6, soluble IL-6 receptor and gp130[1,42]. This implicatestrans-signaling as a possible contributor to discogenic back pain andmay indicate the IL-6/sIL-6R/gp130 complex as a primary target fortherapeutic development for the treatment of discogenic back pain.

IL-6 induced sensitization in peripheral neuropathy and arthritis modelshas been observed due to increased phosphorylation of heat-sensitive ionchannels, which causes channel activation at lower temperatures, anincreased expression of heat-sensitive ion channels in sensitizedneurons, or a combination of the two mechanisms. TRPV1 is a heatsensitive ion channel[12] expressed in nociceptive DRG neurons[6] thathas demonstrated involvement in IL-6 induced thermal hyperalgesia inneuropathy models and can be sensitized via phosphorylation by ProteinKinase A (PKA) or Protein Kinase C (PKC)[22,23]. The phosphorylation ofTRPV1 by PKA/PKC is regulated by AKAP 150/79 (79 is human and 150 is therodent analog)[22,23], a scaffolding protein, that is co-expressed andco-localized with TRPV1 in DRG neurons [8,22] and mediates TRPV1phosphorylation by facilitating interactions between PKA/PKC and TRPV1.As a result, the role of TRPV1 sensitization was investigated viaphosphorylation by inhibiting AKAP 150 via the inhibitor peptideSt-HT31[23]. When blocking the PKA/PKC binding site on AKAP 150 with theinhibitor peptide St-HT31, the observed neuronal thermal sensitivityinduced by degenerative IVD was abolished. These results indicated thatAKAP150/TRPV1 interaction was required for thermal sensitization ofneurons and was the primary mechanism for IL-6 induced sensitization bydegenerative IVD. Additionally, the abolition of IL-6 induced thermalsensitization by inhibiting AKAP 150/TRPV1 interaction establishes AKAP150/79 as a potential therapeutic target in discogenic back pain.

The results show that lentiviral CRISPR epigenome editing of nociceptiveneurons inhibits degenerative IVD sensitization to noxious stimuli,particularly heating. CRISPR based epigenome editing allows for thelocal, long-term, stable site directed gene repression by H3K9 histonemethylation. It was hypothesized that degenerative IVD inducedsensitization of neurons could be regulated by epigenome editing painsensitization pathway genes in nociceptive neurons. The data demonstratethat sensory neurons with epigenome modification of AKAP 150 expressionin sensory neurons maintained a normal firing response under conditionsthat sensitize non-edited neurons to noxious stimuli. These resultsdemonstrate epigenome editing of pain related genes in nociceptiveneurons regulates sensitization and establishes epigenome modificationof pain pathway genes in nociceptive neurons as a therapeutic strategyfor discogenic back pain treatment.

In this study, it was demonstrated that pH, at degenerative IVD levels,and degenerative IVD released factors (IL-6) synergistically sensitizeTRPV1 channels to fire at sub 38° C. temperatures through theIL6/AKAP/TRPV1 signaling pathway, which would provide a mechanism fornociceptor firing in the degenerative IVD environment. In addition, itwas demonstrated that epigenome editing of AKAP expression in sensoryneurons prevented degenerative disc environment triggered neuronsensitization to noxious stimuli. These results elucidate a therapeutictarget and establish epigenome editing of nociceptive neurons as atreatment strategy for discogenic back pain.

Example 2: CRISPR Based Epigenome Editing of Inflammatory Receptors forthe Promotion of Cell Survival and Tissue Deposition in InflammatoryEnvironments

Materials and Methods

Plasmids, Guide Design and Cloning.

Using the UCSC genome browser (GRCh37/hg19), the 5-UTR and the promoterregion, 1000 base pairs upstream were selected and input into the CRISPRdesign tool (crispr.mit.edu). This tool outputs 20 bp gRNAs that arefollowed on their 3′ end by the PAM sequence NGG, which is specific tothe CRISPR-Cas9 system derived from Streptococcus Pyogenes.Additionally, the CRISPR design tool scores the potential gRNA sequencesbased on the number of off-target sites they have and how many arewithin genes (e.g., higher score means less off-target sites). From thislist, the highest scoring gRNAs were input into the BLAT tool of theUCSC genome browser to inspect for overlap of gRNAs with DNAsehypersensitivity peaks, therefore narrowing down potential gRNAs. Withthese criteria, 5-6 gRNAs were selected for TNFR1 and IL1R1 (Table 3.These selected gRNAs were synthesized (University of Utah peptidesynthesis core), annealed, phosphorylated and ligated into hU6-gRNAplasmids at the BbsI sites. For each gene two high performing U6-gRNAcassettes were PCR amplified and transferred into the S. Pyogenes dCas9expressing repression lentiviral vector (hUbC-dCas9-KRAB-T2A-GFP)upstream of the hUbC promoter at the Esp3I sites. An NF-kB reporterplasmid was obtained from Addgene (49343) for experiments assaying NF-kBactivity.

TABLE 3 Target sequences for each gene and the control. Target SEQ IDGene Name Sequence NO. TNFR1 gRNA 1 5′-CAGTGTTGCAACAGCGGGAC-3′  1 TNFR1gRNA 2 5′-AGACTCGGGCATAGAGATCA-3′  2 TNFR1 gRNA 35′-GAATGGCAGGCACCCAGTCA-3′  3 TNFR1 gRNA 4 5′-ATAAGCGTCCGACACATGAT-3′  4TNFR1 gRNA 5 5′-GCAAGGGGCTTATTGCCCCT-3′  5 IL1R1 gRNA 15′-GGAGTCGCCAACTCAATTCG-3′  6 IL1R1 gRNA 2 5′-TGGGGTCCTTGGGCGACTGC-3′  7IL1R1 gRNA 3 5′-AGAGGCATTTCCCGGACTCG-3′  8 IL1R1 gRNA 45′-AGCACAAAGTTGGCTGCGCC-3′  9 IL1R1 gRNA 5 5′-GGGAGGTGACACCCAGTTTA-3′ 10IL1R1 gRNA 6 5′-AAAGTAGCCTGACGTATCCG-3′ 11 N/A Nontarget5′-TTTTTAATACAAGGTAATCT-3′ 12 gRNA

TABLE 4 Guide RNA sequences for each gene and the control Target SEQ IDGene Name Sequence NO. TNFR1 gRNA 1 5′-CAGUGUUGCAACAGCGGGAC-3′ 79 TNFR1gRNA 2 5′-AGACUCGGGCAUAGAGAUCA-3′ 80 TNFR1 gRNA 35′-GAAUGGCAGGCACCCAGUCA-3′ 81 TNFR1 gRNA 4 5′-AUAAGCGUCCGACACAUGAU-3′ 82TNFR1 gRNA 5 5′-GCAAGGGGCUUAUUGCCCCU-3′ 83 IL1R1 gRNA 15′-GGAGUCGCCAACUCAAUUCG-3′ 84 IL1R1 gRNA 2 5′-UGGGGUCCUUGGGCGACUGC-3′ 85IL1R1 gRNA 3 5′-AGAGGCAUUUCCCGGACUCG-3′ 86 IL1R1 gRNA 45′-AGCACAAAGUUGGCUGCGCC-3′ 87 IL1R1 gRNA 5 5′-GGGAGGUGACACCCAGUUUA-3′ 88IL1R1 gRNA 6 5′-AAAGUAGCCUGACGUAUCCG-3′ 89 N/A Nontarget5′-UUUUUAAUACAAGGUAAUCU-3′ 90 gRNA

Cell Culture.

HEK293T cells and immortalized hADMSCs were obtained from the AmericanTissue Collection Center (ATCC). HEK293T cells were cultured in highglucose DMEM (Gibco) supplemented with 10% FBS (Hyclone) and 5 μg/mLgentamicin and subcultured according to manufacturer's instructions.Human ADMSCs were cultured in mesenchymal stem cell basal medium (ATCC,PCS-500-300), supplemented with the ADMSC growth kit (ATCC, PCS-500-40)and 25 μg/mL gentamicin and subcultured according to manufacturer'sinstructions. All cell cultures were maintained at 37° C. with 5% CO₂with media changed every 2-3 days.

Lentivirus Production.

Production of lentivirus was performed in HEK293T cells. Prior toplating cells, 6-well culture dishes were pretreated with 0.001%poly-1-lysine for one hour, rinsed 2× with PBS and air dried. Oncepretreated dishes were ready, HEK293T cells were plated at a density of600,000 cells/well in high glucose DMEM (Gibco) supplemented with 10%FBS in 6-well plates. The next day cells were cotransfected with 4 μg ofthe appropriate lentiviral vector, 3 μg of the packaging plasmid psPAX2(Addgene, 12260), and 1.2 μg of the envelope plasmid pMD2.G (Addgene,12259) using lipofectamine 2000. After 16-20 hours the transfectionmedium was removed and replaced with 2 mL of fresh medium. Supernatantcontaining lentivirus was collected 24 and 48 hours after removal oftransfection medium and filtered through a 0.45 μm PVDF filter. Viruswas immediately aliquoted and frozen at −80° C. after filtration untiluse.

Measurement of Regulation of Gene Expression.

Production of Stable Cell Lines.

Cells were plated at a density of either 400,000 cells/well (HEK293Tcells) or 200,000 cells/well (hADMSCs) in 6-well plates in respectiveexpansion media and allowed to adhere overnight. The next day 1 mL oflentivirus, supplemented with polybrene at a concentration of 8 μg/mL,was placed on cells. After 24 hours virus was removed and washed withPBS 5 times to remove remains of lentivirus. Cells were cultured orfrozen down until use. Regarding dCas9-KRAB expressing cells, cells weresorted via flow cytometry using a FACS Aria Cell Sorter to select forGFP positive cells. Cells were cultured or frozen down until use.

Quantitative Reverse Transcriptase PCR.

Total RNA was harvested from cells using Purelink RNA mini kit (Ambion).Purified RNA subsequently digested with amplification grade DNAse I(applied biosystems) according to the manufacturer's protocol and thencDNA synthesis was performed with DNAse I digested RNA using theHigh-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor(Applied Biosystems, 4374966). Following cDNA synthesis qPCR was formedusing Taqman gene expression assays using best coverage primers for eachgene (Hs01042313_m1, Hs00991002_m1). Changes in gene expression wereexpressed as fold changes in mRNA expression relative to the nontargetcontrol, and normalized to GAPDH mRNA expression, using the ΔΔC_(t)method.

Guide RNA Screening in HEK293T Cells.

HEK293T cells stably expressing dCas9-KRAB GFP were seeded in 6-wellplates at a density of 600,000 cells/well in DMEM-HG with 10% FBS. Thefollowing day 2 μg of each gRNA plasmid (one gRNA per well, n=3) wastransfected into the cells using lipofectamine 2000 according tomanufacturer's instructions. Forty-eight hours post transfection RNA wasisolated, DNase I digested and used for cDNA synthesis. Subsequentlyeach sample was then run through taqman qPCR using primers for TNFRSF1Aor IL1R1 and housekeeping gene GAPDH to quantify downregulation of eachgene by each guide. Gene downregulation was quantified using the ΔΔCtmethod to characterize fold change relative non-target gRNA expressingcells.

Measurement of Functional Effects of Epigenome Editing Based GeneDownregulation.

NF-κB Activity Assays.

HEK293T cells and hADMSCs were transduced with the earlier mentionedNF-κB reporter vector and analyzed for changes in NF-κB activity inresponse to dosing cells with TNF-α/IL-1β (RnD systems, ProSpec). Tofurther describe, cells expressing the NF-κB reporter were plated inwhite 96-well plates (HEK293T cells at 25,000 cells/well and hADMSCs at5000 cells/well) and allowed to attach overnight. The next day cellswere dosed with the appropriate amounts of inflammatory cytokines (0,150, 1, and/or 10 ng/mL of TNF-α/IL-1β for 24 hours. At 24 hoursluminescence corresponding to NF-κB activity was measured in one set ofcells using the bright glo assay (Promega) and cell number was measuredon another set of cells under the same treatment using the real-time gloassay (Promega). Changes in NF-κB activity were quantified as a foldchange in luminescence relative to the no dose control for each celltype, with NF-κB activity normalized to cell number.

Three Dimensional Culture Under Inflammatory Conditions.

Human ADMSCs were cultured in pellet cultures within the presence ofTNF-α/IL-1β to observe matrix production and cell proliferation underinflammatory conditions. Three dimensional culture media consisted ofDMEM-HG (thermofisher), insulin (5 μg/mL), transferrin (5 μg/mL),selenous acid (5 ng/mL), 1.25 mg/mL BSA, 0.17 mM ascorbic acid2-phosphate, 0.35 mM proline, 0.1 μm dexamethasone, 10 ng/mL TGFβ-3(ProSpec) and 1% antibiotic/antimycotic (unless specified all reagentswere purchased from Sigma). To create cell pellets, cells weretrysinized, resuspended at a density of 1×10⁶ cells/mL, and 200 μLaliquots were placed in single wells within a polypropylene v-bottomplate and spun at 1000 g for five minutes to create pellets at thebottom of each well. Pellets formed and lifted after 24 hours at whichmedia was replaced and dosing with TNF-α/IL-1β was begun. Pellets werecultured for 28 days with media changed every 3 days after which pelletswere either fixed in 10% NBF or frozen at −80° C. until analysis.

Size Analysis.

Prior to pulling pellets for analysis, pellets were imaged while stillin wells. Images were used to perform cross sectional area analysis ofpellets in ImageJ (https://imagej.nih.gov/ij/). Cross sectional areameasurements were converted from pixel to mm² using a reference objectof known size.

Chondrogenesis Under Inflammatory Conditions.

Human ADMSCs were differentiated in pellet cultures dosed with TNF-α orIL-1β to investigate ability to undergo chondrogenesis underinflammatory conditions. Basal chondrogenic media consisted of DMEM-HG(thermofisher), 1×ITS+ premix (Corning), 0.1 μM dexamethasone, 0.17 mM,0.35 mM ascorbic acid 2-phosphate, 1× antibiotic antimycotic solution(unless specified all reagents were purchased from Sigma). To createcomplete differentiation media, basal chondrogenic media wassupplemented with 10 ng/mL of TGFβ-3 and BMP-6 (Peprotech). To createchondrogenic pellets, cells were trysinized, resuspended in basalchondrogenic media at density of 1.25×10⁶ cells/mL and 200 μL aliquots(250,000 cells) were placed in single wells within a polypropylenev-bottom plate (Corning) and spun at 1000 g for five minutes to createpellets at the bottom of each well. Pellets formed and lifted after 24hours after which media was replaced with complete chondrogenic mediawith added TNF-α/IL-1β to respective wells. Pellets were cultured for 21days with media replaced every 3 days after which pellets were eitherfixed in 10% NBF or lyophilized and frozen at −80° C. until analysis.Spent media was collected at each media change for analysis ofglycosaminoglycan (GAG) content released.

DNA and Glycosaminoglycan Quantification.

Pellet cultures were analyzed for DNA and GAG content. Prior to analysissamples were papain digested at 60° C. for 12-16 hours in 0.125 μg/mLpapain in 0.10 M Na₂HPO₄, 0.010 M Na₂EDTA, and 0.01 M L-cysteinehydrochloride (pH 6.5). DNA content was then measured using the Hoechst33258 assay. Briefly, digested sample was added to Hoechst dye solution(0.1 μg/mL of Hoechst 33258 in 10 mM Tris, 1 mM disodium EDTA, and 0.1mM NaCl at pH 7.4) and fluorescence was measured on a Biotek Synergy HTXplate reader. DNA concentration was based on standard curves made usingcalf thymus DNA (Sigma). Glycosaminoglycan content of the papaindigested pellets was analyzed using the DMMB assay as previouslydescribed (Zheng and Levenston, 2015). Briefly samples were added toDMMB solution (pH 1.5) and the absorbance was measured at 525 and 595 nmand the OD₅₂₅₋₅₉₅ was used to determine concentration. Concentration wasbased on standard curves were made using shark chondroitin-6-sulfate(Sigma).

Histology.

Samples were fixed in 10% NBF for 48 hours and then transferred to 70%EtOH and taken to the University of Utah histology core for furtherprocessing and embedding. Samples were paraffin embedded and 5 μmsections were mounted onto charged slides. Samples were rehydrated andstained with H&E to characterize matrix production. Slides were imagedusing light microscopy (Nikon Eclipse E400, Olympus UC50 Camera) tovisualize sample variability under multiple conditions.

Characterization of Immunomodulatory Properties of Engineered ADMSCs.

To investigate immunomodulatory properties of engineered hADMSC lines,their suppressive effect on phytohemagglutinin (PHA) activated PBMCs inco-culture was characterized. Prior to co-culture hADMSCs were MitomycinC treated (10 μg/mL) for 2 hours at 37 C. After treatment hADMSCs wererinsed 2× with PBS, trypsinized and plated at a density of 12,800cells/well in a 96-well plate in RPMI1640 media (thermofisher)supplemented with 10% FBS and 100 U/mL penicillin, and 0.1 mg/mLstreptomycin. The following day PBMCs (ATCC) were added at a density of102,400 cells/well (1:8 hADMSC:PBMC ratio) and active with 5 μg/mL PHA.After two days of coculture EdU was added at a concentration of 10 μMand after 18 hours PBMCs were harvested for analysis. To analyzeproliferation PBMCs were CD45 stained (BD biosciences) and EdU labeledwith the Click-iT EdU flow cytometry kit (thermofisher) according tomanufacturer's instructions. The amount of EdU positive CD45 labeledPBMCs were characterized by flow cytometry to provide a quantitativemeasurement of proliferation compared to the control of PBMCs culturedalone.

Statistical Analysis.

Experiments characterizing efficiency of gRNAs in downregulating geneexpression were analyzed by one-way analysis of variance (ANOVA) withTukey's post hoc test, treating different gRNAs as factors. Experimentslooking at changes in NF-κB activity, pellet size, DNA content and GAGcontent were analyzed by two-way ANOVA with Tukey's post hoc test,treating different cell types and cytokine dose as factors. Experimentsinvestigating immunosuppressive effects were analyzed by one-wayanalysis of variance (ANOVA) on repeated measures with Tukey's post hoctest, treating different types of hADMSCs as factors.

Results

Epigenome Editing of TNFR1 in HEK293T Cells Efficiently DownregulatesNF-κB Activity.

Designed gRNAs were initially tested in HEK293T cells to screen for themost efficient gRNAs and provide proof of concept in signalingregulation. Screening of 5-6 gRNAs for both TNFR1 and IL1R1 demonstratedefficient gene downregulation by multiple gRNAs (FIG. 7) with up to 87%gene downregulation by gRNA 1 for TNFR1 and 67% gene downregulation bygRNA 1 for IL1R1. Downregulation of TNFR1 signaling by gRNA 1, measuredafter 24 hours of dosing with 0, 0.15, 1, and 10 ng/mL of TNF-αdemonstrated effective inhibition of TNFR1 signaling. This was noted bysignificant decreases in NF-κB activity (up to 95%) with no significantincrease in NF-κB activity at the lowest most physiologically relevantdose of 0.15 ng/mL.

Epigenome Editing of TNFR1 and IL1R1 in hADMSCs Downregulates NF-κBActivity.

Testing for gene downregulation by two gRNAs, verified to be efficientin HEK293T cells, in hADMSCs demonstrated up to 90% and 88% genedownregulation of TNFR1 and IL1R1, respectively, thus, demonstratingefficient epigenome editing across multiple cell types (FIG. 8). Ofnote, efficiency by gRNAs flipped for IL1R1 in comparison to data shownin HEK293T cells; therefore, demonstrating how small genetic differencesin the promoter sites can have a large effect on the efficacy of generegulation.

Measurement of NF-κB activity post cytokine dosing in hADMSCsdemonstrated potent downregulation of TNFR1 signaling, with nosignificant increase in NF-κB activity noted at both doses of TNF-α(FIG. 8). Downregulation in signaling of IL1R1 was present but not aspotent, while significant NF-κB activity was decreased at the 0.15 ng/mLdose of IL-1β (40% decrease, FIG. 8). Overall regulation of signalingwas present in these cells, therefore, demonstrating regulation of TNFR1and IL1R1 at signaling level and not just at the gene level.

TNFR1/IL1R1 Epigenome Editing Protects hADMSCs from an InflammatoryEnvironment.

Three dimensional culture of hADMSCs in the presence of TNF-α and IL-1β,demonstrated overall cell protection when the cytokine's respectivereceptor was downregulated. Measurement of cell pellet cross sectionalarea demonstrated the maintenance of size in TNFR1/IL1R1 edited vsnon-target edit cells, therefore, indicating protection from matrixbreakdown (FIG. 9). H&E staining of cell pellets further demonstratedthis with lighter background staining in non-target edit control hADMSCspellets dosed with IL-1β/TNF-α (FIG. 9). Additionally, examining DNAcontent relative to control, TNFR1 edited cells demonstratedsignificantly improved maintenance of DNA content compared to nontargetedit cells (FIG. 9).

When induced to undergo chondrogenic differentiation by TGFβ3 and BMP-6,both edited and unedited cells underwent chondrogenic differentiation asnoted by significant amounts of measured GAG content. When dosed witheither TNF-α or IL-1β in chondrogenic media, chondrogenesis wasinhibited in naïve and non-target edit control hADMSCs as noted bysignificant decreases in GAG content relative to undosed controls (FIG.10). Looking specifically at GAG/pellet, significant decreases were seenin all samples but the decrease was less in TNFR1 edited cells. Whenobserving GAG within spent media TNFR1 edited cells did not show asignificant decrease in GAG released while all other cell types did.Observing total GAG content, TNFR1 edited cells showed no significantdecrease in overall GAG content whereas all other cell types did.Therefore this data indicates that epigeneome editing baseddownregulation TNFR1 expression allows hADMSCs to differentiate in anenvironment with elevated levels of TNF-α while IL1R1 downregulationstill allows IL-1β to have an inhibitory effect on chondrogenesis.

Immunomodulatory Properties of hADMSCs are Maintained Post EpigenomeEditing.

Co-culture of hADMSCs with PBMCs demonstrated that therapeuticimmunomodulatory properties of these stem cells are maintained afterepigenome editing (FIG. 11). Relative to naïve untransduced hADMSCs,PBMCs cultured with TNFR1 edited hADMSCs showed no significant increasein proliferation therefore indicating that immunomodulatory propertiesare well maintained. PBMCs cultured with IL1R1 edited hADMSCs showed aslight but significant decrease in suppression of PBMC proliferationtherefore indicating a decrease in immunomodulation. Overall, there wasstill a 40% decrease in PBMC proliferation by IL1R1 edited hADMSCsrelative to PBMCs alone; therefore, beneficial immunomodulatoryproperties are still present.

Example 3: Investigating CRISPi Cell-Engineering Methods for Treatmentof Intervertebral Disc Degeneration

Methods

CRISPRi Vector Design.

Sequences for gRNAs for the TNFR1 and ILIR1 promoter region of each genewere selected along with an adjacent protospacer adjacent motif(crispr.mit.edu). A non-targeting gRNA was chosen as a control. EachgRNA was cloned into a plasmid under the control of the U6 promoter. Toscreen the gRNAs for the best knockdown, gRNA plasmids were transfectedinto a Human Embryonic Kidney (HEK293T) cell line stably expressingdCas9-KRAB (Lipofectamine 2000, n=3 per guide). Forty-eight hourspost-transfection, RNA was isolated and quantitative reversetranscriptase PCR (qRT-PCR) was performed for TNFR1, IL1R1, and GAPDH.For TNFRI, the gRNA showing the greatest knockdown was cloned into twodifferent lentiviral vectors, resulting in the co-expression of the gRNAwith dCas9-T2A-GFP or dCas9-KRAB-T2AGFP from a single lentiviral vector.

Human NP Cell CRISPRi.

Human nucleus pulposus (NP) cells were obtained from a 51-year-old malediscectomy patient and transduced with the TNFR1 lentiviral CRISPRivectors along with a control lentiviral vector that did not express agRNA. Quantitative RT-PCR was performed to identify TNFR1 knockdown inthese primary human NP cells. Additionally, flow-cytometry was performedto determine transduction efficiency in human NP cells.

Functional TNFRJ Knockdown.

Functional knockdown of TNFR1 was studied by quantifying TNF-α inducedNF-KB activity after CRISPRi knockdown. NF-KB activity was quantifiedusing a firefly luciferase NF-KB reporter vector that was transducedinto HEK293T cells [5]. NF-KB reporter expressing HEK293T cells werealso transduced with the developed TNFRI lentiviral CRISPRi vector andcell sorted for GFP to produce a cell line expressing the TNFR1 CRISPRisystem. These cells along with the non-knockdown cells were seeded at25,000 cells/well in white opaque plates and treated with either 0ng/ml, 150 pg/ml, 1 ng/ml, and 10 ng/ml rhTNF-α, for 24 hours (n=5 pertreatment). NF-KB activity was then measured using a firefly luciferaseluminescence assay (Bright-Glo, Promega). Additionally, cell number wasassayed (RealTime-Glo, Promega) on a separate plate with the sameexperimental conditions, therefore allowing for normalization of eachNF-KB luminescence signal to the number of viable cells. Luminescencefor both assays was measured using a synergy HTX biotek plate reader.Activity of NF-KB is reported as a fold change relative to control andnormalized by cell viability.

Statistical Analysis.

To determine if knockdown of IL1R1 and TNFR1 in HEK293T cells wasstatistically significant, a one-way ANOVA was conducted with Tukey posthoc. For statistical analysis of NF-KB activity, a two-way ANOVA withTukey post hoc was conducted and a p<0.05 was considered statisticallysignificant.

Results

CRISPRi Vector Design.

Quantitative reverse transcriptase PCR studies for both TNFR1 and IL1R1in HEK293T cells show significant knockdown in four out of five guidesfor TNFR1 and two out of seven guides for IL1R1. Maximum knockdown inthe guides screened was 82% for TNFRI and 67% for IL1R1 (FIG. 12A-B).Using gRNA 4 for TNFR1 with dCas9-KRAB, 55% TNFR1 knockdown was observedin NP cells but no knockdown with dCas9 alone (FIG. 13B). Transductionefficiency of TNFR1 lentiviral CRISPRi vector in human NP cells was 42%.

Functional TNFRI Knockdown.

Fold change in NF-KB activity was significantly decreased in response toTNF-α after TNFR1 CRISPRi knockdown at each TNF-α dose, demonstrating adecrease in TNFR1 induced signaling (FIG. 13). The decrease in activitywas on the order of −95% for the two highest doses and returned thelowest dose to baseline levels. These results indicate a completeelimination of this deleterious signaling pathway at the mostphysiologically relevant dose.

Discussion

These studies show that CRISPRi knockdown of inflammatory receptors canbe used to modulate the inflammatory response relevant to discpathology. These results also show that CRISPRi can be used tosignificantly reduce gene expression of TNFRI and IL1R1 in HEK293T cellsand TNFR1 gene expression in human NP cells (FIGS. 12 and 13). Theresults also show that this measured reduction in TNFR1 gene expressionalso results in a loss of TNFR1 signaling, as demonstrated by thesignificantly lower fold change in NF-KB activity in TNFR1 knockdownversus number of knockdown cells for each TNF-α dose (FIG. 14). Thisfinding relates to disc pathology, as NF-KB signaling has beenidentified as a component in the degenerative process and antagonizingNF-KB activity has shown therapeutic effects in preclinical animalmodels. The data also shows that TNFRI CRISPRi was able to return thephysiologically relevant dose (150 pg/ml) to baseline levels. Despitethe 42% transduction efficiency, the CRISPRi system showed expressionand activity in human primary NP cells, making their application to thedisc a possibility. Methods to improve transduction efficiency in theprimary human NP cells and the ability of this system to promote viabletissue formation in the inflammatory environment in the degenerative IVDcan also be investigated. These data show a step in developing andapplying the CRISPRi dCas9 system to disc pathology, which can be usedfor both gene therapy applications and tissue engineering applicationstargeting DOD.

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What is claimed is:
 1. A CRISPR-Cas system comprising one or morevectors comprising: a) a promoter operably linked to one or morenucleotide sequences encoding a CRISPR-Cas system guide RNA (gRNA),wherein the gRNA hybridizes with a target sequence of a DNA locus in acell; and b) a regulatory element operably linked to a nucleotidesequence encoding a RNA-directed nuclease, wherein components a) and b)are located on the same or different vectors of the same system, whereinthe gRNA targets and hybridizes with the target sequence and directs theRNA-directed nuclease to the DNA locus; wherein the gRNA sequence isselected from the group listed in Table 2 and Table
 4. 2. The CRISPR-Cassystem of claim 1, wherein the RNA-directed nuclease is a dCas9 protein.3. The CRISPR-Cas system of claim 2, wherein the Cas9 protein is codonoptimized for expression in the cell.
 4. The CRISPR-Cas system of claim1, wherein the cell is a eukaryotic cell.
 5. The CRISPR-Cas system ofclaim 1, wherein the cell is a mammalian or human cell; and/or amesenchymal stem cell.
 6. The CRISPR-Cas system of claim 1, wherein theexpression of one or more gene products is decreased.
 7. The CRISPR-Cassystem of claim 1, wherein the promoter is H1 or U6.
 8. The CRISPR-Cassystem of claim 1, wherein the regulatory element is hUbC.
 9. TheCRISPR-Cas system of claim 1, wherein the system is packaged into asingle lentiviral, adenoviral or adeno-associated virus particle.
 10. Avector comprising: a) a promoter operably linked to one or morenucleotide sequences encoding a CRISPR-Cas system guide RNA (gRNA; andb) a regulatory element operably linked to a nucleotide sequenceencoding a RNA-directed nuclease; wherein the gRNA sequence is selectedfrom the group listed in Table and Table 4
 11. The vector of claim 10,wherein components a) and b) are located on the same or differentvectors of the same system.
 12. The vector of claim 10, wherein theRNA-directed nuclease is a dCas9 protein.
 13. The vector of claim 12,wherein the Cas9 protein is codon optimized for expression in the cell.14. The vector of claim 10, wherein the cell is a eukaryotic cell. 15.The vector of claim 10, wherein the cell is a mammalian or human cell;and/or a mesenchymal stem cell.
 16. The vector of claim 10, wherein theexpression of one or more gene products is decreased.
 17. The vector ofclaim 10, wherein the promoter is H1 or U6.
 18. The vector of claim 10,wherein the regulatory element is hUbC.
 19. A method of modulatingexpression of a gene in a cell, the method comprising: a) introducinginto the cell a first nucleic acid encoding a guide RNA comprising aDNA-binding domain, wherein the nucleic acid is operably linked to anregulatory element, wherein the guide RNA is complementary to a targetnucleic acid sequence comprising the gene; b) introducing into the cella second nucleic acid encoding a transcriptional regulator protein ordomain that modulates the target nucleic acid expression, and comprisesan gRNA-binding domain, wherein the second nucleic acid is operablylinked to a regulatory element; and c) introducing into the cell a thirdnucleic acid encoding a deactivated nuclease Cas9 (dCas9) protein,wherein its nuclease function has been removed, wherein the thirdnucleic acid is operably linked to a regulatory element, wherein thedeactivated nuclease Cas9 protein interacts with the guide RNA, and isfused to the transcriptional regulator protein; wherein the cellproduces the guide RNA, which binds the dCas9 protein and thetranscriptional regulator protein or domain fused to the DNA-bindingdomain, and directs the complex to the DNA regulatory element encoded inthe DNA-binding domain; wherein the guide RNA and the dCas9 proteinco-localize to the target nucleic acid sequence and wherein thetranscriptional regulator protein or domain modulates expression of thegene; wherein the gRNA sequence is selected from the group listed inTable 2 and Table.
 20. A method for introducing into a cell a CRISPR-Cassystem comprising one or more vectors, the vector comprising: a) apromoter operably linked to one or more nucleotide sequences encoding aCRISPR-Cas system guide RNA (gRNA), wherein the gRNA hybridizes with atarget sequence of a DNA molecule in a cell; b) a regulatory elementoperably linked to a nucleotide sequence encoding a RNA-directednuclease, wherein components a) and b) are located on the same ordifferent vectors of the same system, wherein the gRNA targets andhybridizes with the target sequence and directs the RNA-directednuclease to the DNA molecule; wherein the gRNA sequence is selected fromthe group listed in Table and Table
 4. 21. A method for introducing intoa cell a vector comprising, the vector comprising: a) a promoteroperably linked to one or more nucleotide sequences encoding aCRISPR-Cas system guide RNA (gRNA); b) a regulatory element operablylinked to a nucleotide sequence encoding a RNA-directed nuclease;wherein the gRNA sequence is selected from the group listed in Table andTable
 4. 22. A pharmaceutical composition comprising the nucleic acidsequence of any of claims 1-18.
 23. The pharmaceutical composition ofclaim 22, wherein the composition comprises a pharmaceuticallyacceptable carrier.
 24. The pharmaceutical composition of claim 23,wherein the pharmaceutically acceptable carrier comprises a lipid-basedor polymer-based colloid.
 25. The pharmaceutical composition of claim24, wherein the colloid is a liposome, a hydrogel, a microparticle, ananoparticle, or a block copolymer micelle.
 26. The pharmaceuticalcomposition of claim 22, wherein the composition is formulated forintervertebral administration.
 27. A method of treating a subject havinglower back pain, the method comprising administering to the subject atherapeutically effective amount of the composition of any of claim 1-18or 22-26.
 28. The method of claim 27, further comprising identifying asubject having lower back pain.
 29. The method of claim 27, wherein inthe lower back pain is caused by degenerative disc disease.
 30. Themethod of claim 27, wherein the composition is administered into oradjacent to the intervertebral disc.
 31. A method of treating a subjecthaving degenerative disc disease, the method comprising: (a) determiningTNF, IL 1, IL6, IL9, IFN-gamma, and/or IL17 levels in the subject; (b)administering to the subject a pharmaceutical composition comprising anucleic acid sequence encoding a CRISPR-associated deactivatedendonuclease and one or more guide RNAs, wherein the guide RNA isselected from the group listed in Table 2 and Table
 4. 32. The method ofclaim 31, wherein the CRISPR-associated endonuclease is optimized forexpression in a human cell.